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ELIZABETH NOLAN:
Today's recitation

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00:00:21,870 --> 00:00:26,910
will all stem from this
reading by Youngman and Green

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00:00:26,910 --> 00:00:28,650
on ribosome purification.

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00:00:28,650 --> 00:00:31,606
But also, we posted
an optional-- well,

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00:00:31,606 --> 00:00:33,480
not really optional but
an additional reading

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00:00:33,480 --> 00:00:38,100
that's a review that talks
about purifying macromolecular

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00:00:38,100 --> 00:00:39,342
machines from the source.

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00:00:39,342 --> 00:00:40,800
And I guess one
thing I'd just like

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00:00:40,800 --> 00:00:43,100
to say about this
review, something

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00:00:43,100 --> 00:00:46,530
I like is figure three
because it indicates

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00:00:46,530 --> 00:00:49,140
many, many different types
of methods that can be used

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00:00:49,140 --> 00:00:51,812
to purify some biomolecule.

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00:00:51,812 --> 00:00:55,030
And I think often it's easy
just to think all the time,

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00:00:55,030 --> 00:00:56,450
well, let's just use a tag.

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00:00:56,450 --> 00:00:58,970
And tags have
enabled many things,

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00:00:58,970 --> 00:01:00,780
but there's also
many possibilities

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00:01:00,780 --> 00:01:03,150
out there and
decades worth of work

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00:01:03,150 --> 00:01:05,310
before tags for how
to purify proteins.

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00:01:05,310 --> 00:01:07,680
And in some instances, you
might not want to use a tag

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00:01:07,680 --> 00:01:10,630
and that's discussed
to some degree here.

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00:01:10,630 --> 00:01:12,780
So I guess I'm curious.

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00:01:12,780 --> 00:01:15,690
What did you all think
about this week's paper?

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00:01:15,690 --> 00:01:16,830
Did you like it?

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00:01:16,830 --> 00:01:17,760
Did you not like it?

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00:01:17,760 --> 00:01:19,710
Was it easy to hard read?

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00:01:27,300 --> 00:01:28,990
Did you read the paper?

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00:01:35,295 --> 00:01:37,235
AUDIENCE: It wasn't too bad.

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00:01:37,235 --> 00:01:38,036
I've purified--

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00:01:38,036 --> 00:01:39,660
JOANNE STUBBE: You
need to speak louder

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00:01:39,660 --> 00:01:40,630
because I can't hear you.

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00:01:40,630 --> 00:01:41,796
AUDIENCE: It wasn't too bad.

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00:01:41,796 --> 00:01:43,898
I've purified
proteins before, so I

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00:01:43,898 --> 00:01:47,090
felt like I could follow along.

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00:01:47,090 --> 00:01:49,820
ELIZABETH NOLAN: So
not too bad means

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00:01:49,820 --> 00:01:53,620
it was easy to understand
and follow the text there.

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00:01:53,620 --> 00:01:57,420
Yeah, so compared to the reading
for recitations two and three,

44
00:01:57,420 --> 00:02:00,420
this one was probably
easier to work with,

45
00:02:00,420 --> 00:02:03,780
but there's a lot of details
in this one too there.

46
00:02:03,780 --> 00:02:06,870
One thing, I guess, I like this
paper from the standpoint of it

47
00:02:06,870 --> 00:02:10,710
being a methods paper,
is the amount of detail

48
00:02:10,710 --> 00:02:14,640
they give with how they did this
purification and things that

49
00:02:14,640 --> 00:02:16,230
didn't work, right?

50
00:02:16,230 --> 00:02:19,980
So often, sharing those
details with your readers

51
00:02:19,980 --> 00:02:23,480
can make such difference
for an experimentalist

52
00:02:23,480 --> 00:02:26,220
in another lab in another
part of the world, right?

53
00:02:26,220 --> 00:02:30,510
And I think, from this, one
with biochemistry training

54
00:02:30,510 --> 00:02:34,420
could go into the lab and
reproduce their purification

55
00:02:34,420 --> 00:02:34,920
there.

56
00:02:34,920 --> 00:02:38,340
So it's a good example in terms
of what details to include

57
00:02:38,340 --> 00:02:41,810
and what types of
pitfalls to include.

58
00:02:41,810 --> 00:02:44,970
So how many of you have
done a protein purification,

59
00:02:44,970 --> 00:02:48,312
either in a lab class
or in your research?

60
00:02:48,312 --> 00:02:49,280
OK.

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00:02:49,280 --> 00:02:52,920
And so what type of
purification did you use?

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00:02:52,920 --> 00:02:56,647
AUDIENCE: We used a
histamine-tag with nickel.

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00:02:56,647 --> 00:02:57,480
ELIZABETH NOLAN: OK.

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00:02:57,480 --> 00:03:00,720
So you used a His6-tag,
probably a polyhistamine tag

65
00:03:00,720 --> 00:03:02,370
in a nickel NTA column.

66
00:03:02,370 --> 00:03:05,220
For everyone else, has it
been that type of methodology

67
00:03:05,220 --> 00:03:06,450
or another methodology?

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00:03:06,450 --> 00:03:07,845
AUDIENCE: I used the same one.

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00:03:07,845 --> 00:03:09,050
ELIZABETH NOLAN: OK.

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00:03:09,050 --> 00:03:09,550
OK.

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00:03:09,550 --> 00:03:12,750
So would someone like
to kind of comment

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00:03:12,750 --> 00:03:15,890
on the basics of affinity
tag purification?

73
00:03:15,890 --> 00:03:16,950
So how does this work?

74
00:03:21,530 --> 00:03:23,210
And why did you do it?

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00:03:23,210 --> 00:03:25,400
So what are the advantages?

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00:03:31,601 --> 00:03:33,300
AUDIENCE: So you
have a light chain

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00:03:33,300 --> 00:03:36,950
of histamines on
your protein, and you

78
00:03:36,950 --> 00:03:40,680
use nickel, which is
a metal that chelates

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00:03:40,680 --> 00:03:42,640
to histamine very well.

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00:03:47,447 --> 00:03:48,030
Is that right?

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00:03:48,030 --> 00:03:48,540
ELIZABETH NOLAN: Yeah.

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00:03:48,540 --> 00:03:50,164
The nickel's bound
to something though.

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00:03:50,164 --> 00:03:52,125
You have the NTA
ligand on the resin.

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00:03:57,157 --> 00:03:58,698
AUDIENCE: I did this
over a year ago.

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00:03:58,698 --> 00:03:59,670
Sorry.

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00:03:59,670 --> 00:04:02,690
AUDIENCE: So if
you have whatever

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00:04:02,690 --> 00:04:06,810
the type is, the [INAUDIBLE]
bound to some solid substrate.

88
00:04:06,810 --> 00:04:09,300
And then you can
elute everything

89
00:04:09,300 --> 00:04:11,300
that's not bond to that.

90
00:04:11,300 --> 00:04:12,690
Elute what it does bind.

91
00:04:12,690 --> 00:04:13,857
So you just tag the protein.

92
00:04:13,857 --> 00:04:15,273
It's bound to a
high concentration

93
00:04:15,273 --> 00:04:16,107
of the free ligand.

94
00:04:19,110 --> 00:04:20,860
ELIZABETH NOLAN: Or
right, to push it off.

95
00:04:20,860 --> 00:04:25,390
So the idea is that you have
your biomolecule of interest,

96
00:04:25,390 --> 00:04:28,100
whether that be a protein,
which is what we're all

97
00:04:28,100 --> 00:04:31,060
most familiar with,
or the ribosome,

98
00:04:31,060 --> 00:04:32,620
and then you attach a tag.

99
00:04:32,620 --> 00:04:34,900
And that tag can be any
number of things, right?

100
00:04:34,900 --> 00:04:38,050
So in this paper, we saw they
used a stem loop structure

101
00:04:38,050 --> 00:04:41,505
incorporated into the 23s rRNA.

102
00:04:41,505 --> 00:04:42,880
And the idea is
that you're going

103
00:04:42,880 --> 00:04:47,980
to use that tag to separate
your biomolecule of interest

104
00:04:47,980 --> 00:04:51,610
from the complexity of
the cellular environment

105
00:04:51,610 --> 00:04:53,840
there, so all of
the other proteins.

106
00:04:53,840 --> 00:04:56,530
And so you have
some bead or resin

107
00:04:56,530 --> 00:04:58,130
that this tag can bind to.

108
00:04:58,130 --> 00:04:59,680
So in the case of
the nickel column,

109
00:04:59,680 --> 00:05:04,370
the His6-tag will bind to
the nickel NTA on the resin.

110
00:05:04,370 --> 00:05:07,330
And then you can wash
away other components.

111
00:05:07,330 --> 00:05:11,650
And then you devise some
method to elute the protein

112
00:05:11,650 --> 00:05:14,380
you hope to have trapped there.

113
00:05:14,380 --> 00:05:17,740
So what are some advantages
of using an affinity tag,

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00:05:17,740 --> 00:05:19,210
just thinking about
this generally

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00:05:19,210 --> 00:05:20,620
before we delve into the paper?

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00:05:25,380 --> 00:05:27,417
AUDIENCE: Easy to install.

117
00:05:27,417 --> 00:05:28,250
ELIZABETH NOLAN: OK.

118
00:05:28,250 --> 00:05:31,803
So what do you mean
by easy to install?

119
00:05:31,803 --> 00:05:33,775
AUDIENCE: If you
wanted to just encode

120
00:05:33,775 --> 00:05:40,184
a 6 His-tag at the terminus
of your target protein,

121
00:05:40,184 --> 00:05:42,170
I don't know if you
can say it's trivial.

122
00:05:42,170 --> 00:05:43,089
It's easy.

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00:05:43,089 --> 00:05:44,630
ELIZABETH NOLAN:
I'd agree it's easy.

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00:05:44,630 --> 00:05:46,600
There's many plasmids
available that you

125
00:05:46,600 --> 00:05:50,020
can insert your gene of
interest into in order to have

126
00:05:50,020 --> 00:05:51,820
this tag genetically encoded.

127
00:05:51,820 --> 00:05:54,610
So when you express the
protein, the tag's there.

128
00:05:54,610 --> 00:05:58,440
So beyond that, from the
standpoint of purification--

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00:05:58,440 --> 00:06:01,280
AUDIENCE: It's more specific.

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00:06:01,280 --> 00:06:02,280
ELIZABETH NOLAN: Pardon?

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00:06:02,280 --> 00:06:03,400
AUDIENCE: It's more specific.

132
00:06:03,400 --> 00:06:04,733
ELIZABETH NOLAN: More specific--

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00:06:04,733 --> 00:06:08,960
AUDIENCE: Pure proteins as
opposed to various charges.

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00:06:08,960 --> 00:06:11,050
AUDIENCE: It simplifies
the purification.

135
00:06:11,050 --> 00:06:13,976
Instead of doing size
exclusion and ion exchange

136
00:06:13,976 --> 00:06:15,850
that is much different.

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00:06:15,850 --> 00:06:18,790
ELIZABETH NOLAN: So the hope is
it simplifies the purification

138
00:06:18,790 --> 00:06:20,890
because you have
some way initially

139
00:06:20,890 --> 00:06:26,620
to pull your protein of interest
out of your cell lysate there.

140
00:06:26,620 --> 00:06:30,040
So that can be a big help.

141
00:06:30,040 --> 00:06:32,790
What are some potential
disadvantages of using a tag?

142
00:06:32,790 --> 00:06:36,086
So have any of you run into
trouble with a tag in the lab?

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00:06:36,086 --> 00:06:38,882
AUDIENCE: Having a tag can
highly deform your protein

144
00:06:38,882 --> 00:06:39,724
and change it.

145
00:06:39,724 --> 00:06:40,640
ELIZABETH NOLAN: Yeah.

146
00:06:40,640 --> 00:06:43,120
So it might change your
protein or deform it.

147
00:06:43,120 --> 00:06:45,262
What do you mean by "deform?"

148
00:06:45,262 --> 00:06:47,617
AUDIENCE: It could just
cause a conformation change

149
00:06:47,617 --> 00:06:51,251
or the tag could make it
localize somewhere else based

150
00:06:51,251 --> 00:06:52,794
on size.

151
00:06:52,794 --> 00:06:53,710
ELIZABETH NOLAN: Yeah.

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00:06:53,710 --> 00:06:57,520
So that's an example, say if
you were doing a study in cells,

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00:06:57,520 --> 00:06:58,870
say, rather than a purification.

154
00:06:58,870 --> 00:07:00,286
But maybe you tag
a protein and it

155
00:07:00,286 --> 00:07:03,250
goes somewhere other than
it would go untagged right?

156
00:07:03,250 --> 00:07:05,920
And that will affect
your observations

157
00:07:05,920 --> 00:07:08,050
and your data there.

158
00:07:08,050 --> 00:07:09,670
And it might alter
the conformation.

159
00:07:09,670 --> 00:07:11,650
So it might affect
the folding, right?

160
00:07:11,650 --> 00:07:14,660
It might affect the
oligomerization.

161
00:07:14,660 --> 00:07:17,020
His-tags bind metal ions.

162
00:07:17,020 --> 00:07:22,140
So is that a factor
to consider there?

163
00:07:22,140 --> 00:07:26,910
If you have an enzyme, will
the tag affect activity, right?

164
00:07:26,910 --> 00:07:29,020
And these things can be
a positive or a negative.

165
00:07:29,020 --> 00:07:32,500
Sometimes the tag is
helpful in these regards.

166
00:07:32,500 --> 00:07:36,670
You can't get soluble protein
without the tag, right?

167
00:07:36,670 --> 00:07:38,800
And sometimes the reverse.

168
00:07:38,800 --> 00:07:42,190
You decide to express your
protein or biomolecule

169
00:07:42,190 --> 00:07:45,520
with a tag and you find out
you get an aggregate, something

170
00:07:45,520 --> 00:07:47,360
that the protein shouldn't be.

171
00:07:47,360 --> 00:07:51,580
So these are just things to keep
in mind when designing a fusion

172
00:07:51,580 --> 00:07:54,040
protein and thinking
about how you're

173
00:07:54,040 --> 00:07:58,450
going to use an affinity tag
to express your protein there

174
00:07:58,450 --> 00:08:00,820
and purify your protein.

175
00:08:00,820 --> 00:08:03,280
So there's pluses
and minuses, right?

176
00:08:03,280 --> 00:08:06,550
And you can always make the
choice not to use a tag.

177
00:08:06,550 --> 00:08:11,110
So you saw some of that in
the review article, right?

178
00:08:11,110 --> 00:08:13,390
And I guess one other
thing, too, there's

179
00:08:13,390 --> 00:08:16,392
this idea of using the affinity
tag in the affinity column,

180
00:08:16,392 --> 00:08:18,850
which we'll talk about more in
the context of the ribosome,

181
00:08:18,850 --> 00:08:20,650
but is that always enough?

182
00:08:20,650 --> 00:08:24,250
So is the affinity column
alone always enough

183
00:08:24,250 --> 00:08:27,520
to purify your
protein of interest?

184
00:08:38,380 --> 00:08:40,600
So in lab class, that's
where it will end,

185
00:08:40,600 --> 00:08:43,000
because that's an exercise
made for lab class,

186
00:08:43,000 --> 00:08:45,880
and it's your first adventure
into protein purification

187
00:08:45,880 --> 00:08:47,620
for most people, right?

188
00:08:47,620 --> 00:08:49,900
Oftentimes, it's not enough.

189
00:08:49,900 --> 00:08:53,590
That you do enrich what you've
purified with what you want,

190
00:08:53,590 --> 00:08:55,000
but often there's contaminants.

191
00:08:55,000 --> 00:08:57,460
So you actually do move forward
with doing some other type

192
00:08:57,460 --> 00:08:58,360
of purification.

193
00:08:58,360 --> 00:09:02,470
Like Rebecca mentioned, ion
exchange or size exclusion,

194
00:09:02,470 --> 00:09:06,190
those are things you can use
after the affinity purification

195
00:09:06,190 --> 00:09:08,830
there as needed, right?

196
00:09:08,830 --> 00:09:13,144
So contaminations are
something to look out for here.

197
00:09:13,144 --> 00:09:14,810
JOANNE STUBBE: What
other types of steps

198
00:09:14,810 --> 00:09:17,218
do you use for purification
besides columns

199
00:09:17,218 --> 00:09:19,960
and [INAUDIBLE].

200
00:09:19,960 --> 00:09:22,530
Because people have
forgotten all of this.

201
00:09:22,530 --> 00:09:26,081
Everybody uses the tag
and that's the end of it,

202
00:09:26,081 --> 00:09:28,740
and it can be the kiss of death.

203
00:09:28,740 --> 00:09:30,640
What other kind of
fractionation steps?

204
00:09:30,640 --> 00:09:32,780
Do you do any other
fractionation steps?

205
00:09:32,780 --> 00:09:35,050
What is it, 535 or something?

206
00:09:37,606 --> 00:09:38,730
AUDIENCE: [INAUDIBLE] salt.

207
00:09:38,730 --> 00:09:39,640
JOANNE STUBBE: The what?

208
00:09:39,640 --> 00:09:41,056
AUDIENCE: The
[INAUDIBLE] salt. So

209
00:09:41,056 --> 00:09:42,700
some proteins will precipitate.

210
00:09:42,700 --> 00:09:43,687
Some will not.

211
00:09:43,687 --> 00:09:44,520
JOANNE STUBBE: Yeah.

212
00:09:44,520 --> 00:09:46,310
So that's what you
use to precipitate.

213
00:09:46,310 --> 00:09:48,920
So that's a mild method.

214
00:09:48,920 --> 00:09:49,960
It's fast.

215
00:09:49,960 --> 00:09:55,190
It gives you separation on
a fair amount of proteins.

216
00:09:55,190 --> 00:09:57,655
Anybody know what you use?

217
00:09:57,655 --> 00:09:58,780
AUDIENCE: Ammonium sulfate.

218
00:09:58,780 --> 00:10:00,450
JOANNE STUBBE: Yeah,
ammonium sulfate.

219
00:10:00,450 --> 00:10:03,330
And then what's the other
thing that really is important?

220
00:10:03,330 --> 00:10:05,830
What is the other thing you
want to remove from your protein

221
00:10:05,830 --> 00:10:09,822
when you're using this tag
that oftentimes people miss

222
00:10:09,822 --> 00:10:10,975
in the literature?

223
00:10:10,975 --> 00:10:12,850
ELIZABETH NOLAN: Yeah,
we were getting there.

224
00:10:12,850 --> 00:10:15,183
JOANNE STUBBE: There's another
component inside the cell

225
00:10:15,183 --> 00:10:18,751
that you need to get rid of
that you've been talking about.

226
00:10:18,751 --> 00:10:22,202
AUDIENCE: Where one component
has His-tag on the cell,

227
00:10:22,202 --> 00:10:24,522
you remove it.

228
00:10:24,522 --> 00:10:26,146
JOANNE STUBBE: So
you have the protein.

229
00:10:26,146 --> 00:10:28,118
What are the other
components in the cell

230
00:10:28,118 --> 00:10:30,090
that you need to remove?

231
00:10:30,090 --> 00:10:32,062
You can go back and
look at your cartoon

232
00:10:32,062 --> 00:10:34,604
of the inside of the cell.

233
00:10:34,604 --> 00:10:35,520
ELIZABETH NOLAN: Yeah.

234
00:10:35,520 --> 00:10:38,560
So we're getting a little
ahead, but that's totally fine.

235
00:10:38,560 --> 00:10:43,140
So if you're going to
lyse your cell, and then,

236
00:10:43,140 --> 00:10:45,540
imagine your protein's
soluble, right?

237
00:10:45,540 --> 00:10:48,090
So you do a
centrifugation to remove

238
00:10:48,090 --> 00:10:49,500
the insoluble components.

239
00:10:49,500 --> 00:10:51,690
So you have the membrane
and all that debris.

240
00:10:51,690 --> 00:10:53,790
And then you take
your soluble fraction,

241
00:10:53,790 --> 00:10:57,000
and you incubate that
on your column, right?

242
00:10:57,000 --> 00:11:00,367
And then you elute, you
wash, you elute your protein.

243
00:11:00,367 --> 00:11:01,950
What might come out
with your protein?

244
00:11:04,590 --> 00:11:05,650
AUDIENCE: Nucleic acid.

245
00:11:05,650 --> 00:11:06,858
ELIZABETH NOLAN: Yeah, right.

246
00:11:06,858 --> 00:11:10,866
So how do you know if you have
nucleic acid contaminating?

247
00:11:10,866 --> 00:11:12,442
AUDIENCE: The A260.

248
00:11:12,442 --> 00:11:13,400
ELIZABETH NOLAN: Right.

249
00:11:13,400 --> 00:11:16,810
So A260 will give you a
readout of nucleic acids.

250
00:11:16,810 --> 00:11:19,549
A280 is what people typically
look at for their protein

251
00:11:19,549 --> 00:11:21,590
concentration, but you
should look at both so you

252
00:11:21,590 --> 00:11:23,249
know what's in your protein.

253
00:11:23,249 --> 00:11:24,290
You need to look at both.

254
00:11:24,290 --> 00:11:26,282
JOANNE STUBBE: Do
that and adaptively

255
00:11:26,282 --> 00:11:27,740
repeat stuff out
of the literature.

256
00:11:27,740 --> 00:11:30,270
Very frequently, you take
an absorption spectrum,

257
00:11:30,270 --> 00:11:31,460
there's some nucleic acid.

258
00:11:31,460 --> 00:11:33,126
ELIZABETH NOLAN: You
have contamination.

259
00:11:33,126 --> 00:11:34,370
So a lot of ribosome, DNA.

260
00:11:34,370 --> 00:11:36,740
JOANNE STUBBE: If you don't
remember anything else,

261
00:11:36,740 --> 00:11:37,580
it's important.

262
00:11:37,580 --> 00:11:38,840
ELIZABETH NOLAN: Yeah.

263
00:11:38,840 --> 00:11:42,150
So sure, just something
to think about.

264
00:11:42,150 --> 00:11:47,882
So Alex noted, it's easy
to put on this His-tag.

265
00:11:47,882 --> 00:11:50,780
What is actually on
this His-tagged protein?

266
00:11:50,780 --> 00:11:54,077
So is it just the six
histidines are all the same?

267
00:11:54,077 --> 00:11:55,910
When you look in the
literature, and someone

268
00:11:55,910 --> 00:12:02,040
says they put a His-tag on the
N terminus of their protein,

269
00:12:02,040 --> 00:12:03,220
how do you think about that?

270
00:12:10,684 --> 00:12:16,900
So we have some
His6-tag, and then

271
00:12:16,900 --> 00:12:24,050
that tag, say, is attached
to the protein here.

272
00:12:24,050 --> 00:12:25,225
What's going on in between?

273
00:12:28,480 --> 00:12:31,350
AUDIENCE: Maybe a flexible
linker of some kind.

274
00:12:31,350 --> 00:12:32,350
ELIZABETH NOLAN: Pardon?

275
00:12:32,350 --> 00:12:33,280
AUDIENCE: It would be like a--

276
00:12:33,280 --> 00:12:35,140
I don't know-- like a
flexible linker of some kind.

277
00:12:35,140 --> 00:12:35,390
ELIZABETH NOLAN: Yeah.

278
00:12:35,390 --> 00:12:37,340
So maybe some kind
of flexible linker.

279
00:12:37,340 --> 00:12:39,680
And what might
dictate this linker?

280
00:12:47,616 --> 00:12:49,600
AUDIENCE: If you wanted
to remove the His-tag

281
00:12:49,600 --> 00:12:53,452
after you would want to be able
to hydrolyze it or something.

282
00:12:53,452 --> 00:12:54,910
ELIZABETH NOLAN:
Yeah, so maybe you

283
00:12:54,910 --> 00:12:57,220
want to remove your tag
down the road, right?

284
00:12:57,220 --> 00:12:59,770
So a protease is often employed.

285
00:12:59,770 --> 00:13:04,750
So maybe there's a linker, maybe
there's a protease cleavage

286
00:13:04,750 --> 00:13:05,250
site.

287
00:13:09,980 --> 00:13:12,321
OK.

288
00:13:12,321 --> 00:13:12,820
OK.

289
00:13:12,820 --> 00:13:15,840
And we're not going to
talk about cloning really

290
00:13:15,840 --> 00:13:20,650
in this class, but just
thinking back a step,

291
00:13:20,650 --> 00:13:24,970
you need some plasmid DNA
to ultimately get here

292
00:13:24,970 --> 00:13:29,240
that has your gene, right?

293
00:13:29,240 --> 00:13:32,260
And so you'll insert the
gene into the plasmid,

294
00:13:32,260 --> 00:13:34,390
and many commercial
plasmids have something

295
00:13:34,390 --> 00:13:36,260
called multiple cloning site.

296
00:13:36,260 --> 00:13:39,640
And, for instance, if you
want a His-tag or a GST tag,

297
00:13:39,640 --> 00:13:41,650
you'll use some
different plasmid

298
00:13:41,650 --> 00:13:43,720
that has that encoded, right?

299
00:13:43,720 --> 00:13:47,350
And then you make a decision
about how you put your gene in.

300
00:13:47,350 --> 00:13:49,390
And these multiple
cloning sites have

301
00:13:49,390 --> 00:13:51,430
multiple sites for
restriction enzymes

302
00:13:51,430 --> 00:13:54,250
where you can put the
gene in, which means,

303
00:13:54,250 --> 00:13:58,710
even if the same plasmid is
used for 10 different proteins,

304
00:13:58,710 --> 00:14:02,200
what's happening
here can vary a lot

305
00:14:02,200 --> 00:14:05,380
even if you have one protein
and put it in different sites.

306
00:14:05,380 --> 00:14:09,880
So maybe you have a short
linker here because you

307
00:14:09,880 --> 00:14:14,301
used a site like NDE1 or SPE1,
I'm just making those up,

308
00:14:14,301 --> 00:14:14,800
right?

309
00:14:14,800 --> 00:14:17,350
Or maybe you have a
longer region here

310
00:14:17,350 --> 00:14:19,110
between the tag and the protein.

311
00:14:19,110 --> 00:14:21,700
And so it's very
important to go look back

312
00:14:21,700 --> 00:14:24,580
at the map of the plasmid
that was used and ask

313
00:14:24,580 --> 00:14:27,550
where was the gene put in
and what does that mean?

314
00:14:27,550 --> 00:14:30,730
So is this His-tag a 2
kilodalton perturbation.

315
00:14:30,730 --> 00:14:33,940
Is it a 5 kilodalton
perterbation to your protein?

316
00:14:33,940 --> 00:14:39,520
And some of these plasmids have
multiple types of tags, right?

317
00:14:39,520 --> 00:14:42,070
So it might have a
GST tag and a His-tag,

318
00:14:42,070 --> 00:14:45,610
and depending how you put
your gene in, you may have two

319
00:14:45,610 --> 00:14:47,620
or you may have only one, right?

320
00:14:47,620 --> 00:14:50,119
So I'm just pointing out
there's a complexity here.

321
00:14:50,119 --> 00:14:51,910
So when someone just
writes in their paper,

322
00:14:51,910 --> 00:14:53,710
oh, I His-tagged
the protein, you

323
00:14:53,710 --> 00:14:56,740
need to think beyond just
sticking six histamine residues

324
00:14:56,740 --> 00:14:58,480
on the N or C terminus there.

325
00:14:58,480 --> 00:15:00,358
Did you have a question?

326
00:15:00,358 --> 00:15:03,047
AUDIENCE: So does that mean
that's like another step where

327
00:15:03,047 --> 00:15:06,226
they purified the
specific plumbing site,

328
00:15:06,226 --> 00:15:07,693
one that they wanted?

329
00:15:07,693 --> 00:15:11,130
Or does that mean you kind of
roll with the heterogeneous--

330
00:15:11,130 --> 00:15:12,490
ELIZABETH NOLAN: No.

331
00:15:12,490 --> 00:15:16,090
So you put your gene
in particular sites

332
00:15:16,090 --> 00:15:18,260
with this type of
cloning, right?

333
00:15:18,260 --> 00:15:24,280
And so one thing
practically, just say

334
00:15:24,280 --> 00:15:26,100
you want to express
some new protein

335
00:15:26,100 --> 00:15:27,880
and you don't know
much about this.

336
00:15:27,880 --> 00:15:31,120
You might choose to make several
different constructs where

337
00:15:31,120 --> 00:15:33,760
you put the gene
in different sites

338
00:15:33,760 --> 00:15:37,300
or maybe you use primers that
allow you to add some linker

339
00:15:37,300 --> 00:15:39,910
regions because you don't
know what will give you better

340
00:15:39,910 --> 00:15:42,420
solubility and better yield.

341
00:15:42,420 --> 00:15:45,730
And then ultimately,
you pick one.

342
00:15:45,730 --> 00:15:52,760
So, for instance, like for
me, with working with, say,

343
00:15:52,760 --> 00:15:56,650
a His-tag for a protein,
there's plenty of plasmids

344
00:15:56,650 --> 00:16:00,040
available where you can pick
N terminus or C terminus.

345
00:16:00,040 --> 00:16:02,770
So one plasmid to put the
tag on the N terminus.

346
00:16:02,770 --> 00:16:05,530
A different plasmid to put
the tag on the C terminus.

347
00:16:05,530 --> 00:16:08,890
I'll clone the gene into
both and test overexpression

348
00:16:08,890 --> 00:16:11,020
with both and just
see if one's better

349
00:16:11,020 --> 00:16:15,070
than the other in terms of
yield, in terms of solubility

350
00:16:15,070 --> 00:16:16,180
there.

351
00:16:16,180 --> 00:16:17,800
And then you make a call.

352
00:16:17,800 --> 00:16:21,430
Maybe you purify both and see if
there is an effect on behavior,

353
00:16:21,430 --> 00:16:25,670
like oligomerization or
activity if it's an enzyme here.

354
00:16:25,670 --> 00:16:28,436
So if you get into
protein purification,

355
00:16:28,436 --> 00:16:30,310
it's good to talk to
people who have purified

356
00:16:30,310 --> 00:16:33,320
many different proteins
because the strategy is

357
00:16:33,320 --> 00:16:35,300
a little different
for each protein.

358
00:16:35,300 --> 00:16:37,270
And then you just
get more exposure

359
00:16:37,270 --> 00:16:42,810
to all of the possibilities
and troubleshooting there.

360
00:16:42,810 --> 00:16:49,000
So coming to the paper,
what was the big motivation

361
00:16:49,000 --> 00:16:53,920
for developing this method to
have an affinity tag attached

362
00:16:53,920 --> 00:16:57,140
to the ribosome?

363
00:16:57,140 --> 00:16:57,640
Right?

364
00:16:57,640 --> 00:17:01,030
So Youngman and Green went
to quite a bit of effort

365
00:17:01,030 --> 00:17:02,320
to devise this new system.

366
00:17:04,877 --> 00:17:05,960
What was their motivation?

367
00:17:10,055 --> 00:17:19,420
And what really
was the big issue

368
00:17:19,420 --> 00:17:22,882
they were seeking to overcome?

369
00:17:22,882 --> 00:17:26,340
AUDIENCE: They wanted to get
ribosomes with mutations on it.

370
00:17:26,340 --> 00:17:30,084
So they want to synthesize
bacteria ribosomes with it

371
00:17:30,084 --> 00:17:30,964
and purify it.

372
00:17:30,964 --> 00:17:31,880
ELIZABETH NOLAN: Yeah.

373
00:17:31,880 --> 00:17:33,740
So they want a mutant ribosome.

374
00:17:38,270 --> 00:17:40,860
And they want to make
this mutant ribosome

375
00:17:40,860 --> 00:17:47,810
in vivo and then purify.

376
00:17:50,420 --> 00:17:54,750
So what's the complication
with making the mutant ribosome

377
00:17:54,750 --> 00:17:59,948
in vivo that they
seek to overcome here?

378
00:17:59,948 --> 00:18:03,260
AUDIENCE: You have the wild-type
ribosomes in there also.

379
00:18:03,260 --> 00:18:04,729
ELIZABETH NOLAN: So well, right.

380
00:18:04,729 --> 00:18:06,020
That was their decision, right?

381
00:18:06,020 --> 00:18:09,560
They want to express this mutant
ribosome in the background

382
00:18:09,560 --> 00:18:11,000
of the wild-type ribosome.

383
00:18:11,000 --> 00:18:14,048
So why do they want to do that?

384
00:18:14,048 --> 00:18:15,952
AUDIENCE: Because
it could be lethal.

385
00:18:15,952 --> 00:18:20,712
The mutant ribosome, it
would be a toxic mutant.

386
00:18:20,712 --> 00:18:22,910
ELIZABETH NOLAN:
Yeah, toxic mutant.

387
00:18:22,910 --> 00:18:24,806
What do you mean
by "toxic mutant?"

388
00:18:24,806 --> 00:18:28,915
AUDIENCE: If you create
a mutant ribosome, that

389
00:18:28,915 --> 00:18:32,806
was the only way for the cell
to express them, they'd be toxic

390
00:18:32,806 --> 00:18:35,234
and they wouldn't be able
to go through translation.

391
00:18:35,234 --> 00:18:36,150
ELIZABETH NOLAN: Yeah.

392
00:18:36,150 --> 00:18:39,240
So maybe the mutant ribosome
doesn't work very well,

393
00:18:39,240 --> 00:18:43,260
and that ends up being
lethal to the cell there.

394
00:18:43,260 --> 00:18:47,250
So can we imagine
why that might be

395
00:18:47,250 --> 00:18:49,770
an issue for the
types of experiments

396
00:18:49,770 --> 00:18:51,040
we've seen in class?

397
00:18:51,040 --> 00:18:53,370
So if you're
thinking about trying

398
00:18:53,370 --> 00:18:57,690
to understand the catalytic
mechanism in that function,

399
00:18:57,690 --> 00:19:01,620
there's a likelihood the
mutations may dramatically

400
00:19:01,620 --> 00:19:03,180
affect that activity, right?

401
00:19:03,180 --> 00:19:04,770
If you want to put
a point mutation

402
00:19:04,770 --> 00:19:07,060
into the peptidyl
transferase center,

403
00:19:07,060 --> 00:19:09,060
into the decoding
center, that could

404
00:19:09,060 --> 00:19:11,760
be deleterious to
your cell, but it

405
00:19:11,760 --> 00:19:14,700
could be very important
for your mechanistic study.

406
00:19:14,700 --> 00:19:18,240
So they want to avoid
this lethal phenotype.

407
00:19:29,250 --> 00:19:32,810
So what is the
complication in terms

408
00:19:32,810 --> 00:19:39,560
of doing this in the
presence of wild-type

409
00:19:39,560 --> 00:19:42,790
for some sort of measurement?

410
00:19:42,790 --> 00:19:44,381
AUDIENCE: It gets kind of muddy.

411
00:19:44,381 --> 00:19:45,880
ELIZABETH NOLAN:
Gets kind of muddy.

412
00:19:45,880 --> 00:19:46,260
Yeah.

413
00:19:46,260 --> 00:19:47,093
What does that mean?

414
00:19:47,093 --> 00:19:50,583
AUDIENCE: You don't have
a pure mutant in there,

415
00:19:50,583 --> 00:19:56,134
and they're not significantly
different from the wild-type.

416
00:19:56,134 --> 00:19:57,050
ELIZABETH NOLAN: Yeah.

417
00:19:57,050 --> 00:19:59,180
So if you were going to
do a standard ribosome

418
00:19:59,180 --> 00:20:00,350
purification--

419
00:20:00,350 --> 00:20:02,690
because ribosomes have been
purified for many years

420
00:20:02,690 --> 00:20:04,265
without an affinity tag--

421
00:20:04,265 --> 00:20:06,140
you're going to have a
mixture of your mutant

422
00:20:06,140 --> 00:20:07,730
and the wild-type.

423
00:20:07,730 --> 00:20:11,630
And so that has a
strong likelihood

424
00:20:11,630 --> 00:20:14,300
of being a problem for
your analysis, right?

425
00:20:14,300 --> 00:20:16,100
So they gave an
example in this paper

426
00:20:16,100 --> 00:20:17,540
where they actually
made a mixture

427
00:20:17,540 --> 00:20:20,390
and did some analyses,
where you could separate

428
00:20:20,390 --> 00:20:22,290
the wild-type from
mutant activity

429
00:20:22,290 --> 00:20:24,840
but that's not
necessarily the case.

430
00:20:24,840 --> 00:20:28,350
And so, as pointed out,
they're both very large.

431
00:20:28,350 --> 00:20:30,110
They're very similar, right?

432
00:20:30,110 --> 00:20:33,500
There's no good way
to separate a ribosome

433
00:20:33,500 --> 00:20:35,060
with a single-point
mutation, say,

434
00:20:35,060 --> 00:20:38,100
in the peptidyl transferase
center from wild-type.

435
00:20:38,100 --> 00:20:45,110
So let's just imagine you have
a mixture that's predominantly

436
00:20:45,110 --> 00:20:48,050
your mutant ribosome but
you have some background

437
00:20:48,050 --> 00:20:49,820
contamination of wild-type.

438
00:20:49,820 --> 00:20:50,600
Is that an issue?

439
00:20:58,536 --> 00:21:05,580
Say you want to measure rates
of peptide bond formation.

440
00:21:05,580 --> 00:21:07,080
AUDIENCE: It's going
to be an issue.

441
00:21:07,080 --> 00:21:07,996
ELIZABETH NOLAN: Yeah.

442
00:21:07,996 --> 00:21:09,941
So why is it probably
going to be an issue?

443
00:21:09,941 --> 00:21:11,624
AUDIENCE: Because
your mutant might

444
00:21:11,624 --> 00:21:15,232
be a lethal function
because of your background

445
00:21:15,232 --> 00:21:20,094
and resume the function and
[INAUDIBLE] mutant [INAUDIBLE]..

446
00:21:20,094 --> 00:21:21,010
ELIZABETH NOLAN: Yeah.

447
00:21:21,010 --> 00:21:23,010
So imagine you have a
mutant ribosome that

448
00:21:23,010 --> 00:21:27,660
has very low activity
or none, and you

449
00:21:27,660 --> 00:21:30,030
have some small amount of
contaminating wild-type that

450
00:21:30,030 --> 00:21:32,610
has wild-type activity, right?

451
00:21:32,610 --> 00:21:34,200
How do you know--

452
00:21:34,200 --> 00:21:36,420
I mean you might
misinterpret your data,

453
00:21:36,420 --> 00:21:39,330
and what you're seeing is the
wild-type and not the mutant.

454
00:21:39,330 --> 00:21:41,880
And this issue is much more
broad than the ribosome.

455
00:21:41,880 --> 00:21:45,240
So one of my favorites is
the contaminating ATPase.

456
00:21:45,240 --> 00:21:48,810
And maybe you have an
enzyme that hydrolyzes ATP,

457
00:21:48,810 --> 00:21:50,640
but maybe you have a
small contamination

458
00:21:50,640 --> 00:21:54,870
of an enzyme that does a much
better job at hydrolyzing ATP

459
00:21:54,870 --> 00:21:56,670
that's in your reaction, right?

460
00:21:56,670 --> 00:21:59,400
So what are you seeing there?

461
00:21:59,400 --> 00:22:01,140
So that's something
to keep in mind

462
00:22:01,140 --> 00:22:04,920
in terms of potential
contaminations, right?

463
00:22:04,920 --> 00:22:06,900
And so they go through
some justification

464
00:22:06,900 --> 00:22:09,270
about why they need to
do this method based

465
00:22:09,270 --> 00:22:13,020
on available methods, and all
of those available methods

466
00:22:13,020 --> 00:22:14,410
have strengths and weaknesses.

467
00:22:14,410 --> 00:22:16,080
So they talked
about using systems

468
00:22:16,080 --> 00:22:18,260
without the wild-type ribosome.

469
00:22:18,260 --> 00:22:20,250
They mentioned in
vitro translation,

470
00:22:20,250 --> 00:22:25,950
and I'll just say, in passing,
those in vitro systems

471
00:22:25,950 --> 00:22:29,861
have improved a lot since
the time of this paper.

472
00:22:29,861 --> 00:22:30,360
OK.

473
00:22:30,360 --> 00:22:33,520
So in terms of their strategy.

474
00:22:33,520 --> 00:22:40,010
Let's comment on the various
aspects of this strategy.

475
00:22:40,010 --> 00:22:40,510
All right.

476
00:22:40,510 --> 00:22:43,650
So effectively, they
want a way to express

477
00:22:43,650 --> 00:22:48,150
the mutant ribosome in vivo in
the background of wild-type.

478
00:22:48,150 --> 00:22:50,730
They want a way to
separate that ribosome.

479
00:22:50,730 --> 00:22:53,951
And they want to come up with
ribosomes that are active.

480
00:22:53,951 --> 00:22:54,450
OK.

481
00:22:54,450 --> 00:22:58,800
So this cartoon basically
summarizes their solution

482
00:22:58,800 --> 00:23:01,920
to this problem,
and we should work

483
00:23:01,920 --> 00:23:06,010
through the various components.

484
00:23:06,010 --> 00:23:09,450
So the first thing is
they attached a tag

485
00:23:09,450 --> 00:23:12,570
to either the 23S or the 16S.

486
00:23:12,570 --> 00:23:15,457
And we'll focus today's
discussion on the 23S

487
00:23:15,457 --> 00:23:17,540
because that's what they
did more characterization

488
00:23:17,540 --> 00:23:18,990
on in the paper.

489
00:23:18,990 --> 00:23:23,000
So how did they decide on the
tag and where to place the tag?

490
00:23:27,930 --> 00:23:29,902
AUDIENCE: You don't
want the tag somewhere

491
00:23:29,902 --> 00:23:32,367
that's going to
interfere with function.

492
00:23:32,367 --> 00:23:34,832
And you don't want the
tag itself to be reactive

493
00:23:34,832 --> 00:23:36,311
where it will interfere.

494
00:23:36,311 --> 00:23:41,241
So you need to be out of
the way and kind of passive

495
00:23:41,241 --> 00:23:43,127
so it's not interfering
with function.

496
00:23:43,127 --> 00:23:44,960
ELIZABETH NOLAN: So
that's one point, right?

497
00:23:44,960 --> 00:23:48,660
We don't want this tag to
interfere with function.

498
00:23:48,660 --> 00:23:50,970
So that's one aspect.

499
00:23:50,970 --> 00:23:52,752
What's another aspect?

500
00:23:52,752 --> 00:23:54,460
AUDIENCE: You still
have to be accessible

501
00:23:54,460 --> 00:23:55,960
ELIZABETH NOLAN:
Yeah, the tag needs

502
00:23:55,960 --> 00:23:57,510
to be accessible
because something's

503
00:23:57,510 --> 00:24:00,350
going to have to
bind this tag, right?

504
00:24:00,350 --> 00:24:04,220
So on the basis of
those two criteria,

505
00:24:04,220 --> 00:24:07,350
we can imagine wanting this
tag somewhere on the surface,

506
00:24:07,350 --> 00:24:07,850
right?

507
00:24:07,850 --> 00:24:11,030
We don't want it where
the 30S and 50S interact.

508
00:24:11,030 --> 00:24:13,820
We don't want it in a
position that's critical.

509
00:24:13,820 --> 00:24:16,330
So like maybe if it
ended up near where

510
00:24:16,330 --> 00:24:19,350
EFTU first binds or
EFG, that would be bad.

511
00:24:19,350 --> 00:24:23,630
So accessible and in a place
where it won't interfere.

512
00:24:23,630 --> 00:24:26,510
Beyond that, the
ribosome's huge.

513
00:24:26,510 --> 00:24:29,360
So how does one pick
where to put this tag?

514
00:24:29,360 --> 00:24:30,650
What did the researchers do?

515
00:24:37,134 --> 00:24:38,550
I'll tell you what
they didn't do.

516
00:24:38,550 --> 00:24:40,240
They didn't reinvent the wheel.

517
00:24:40,240 --> 00:24:43,560
So what did they do?

518
00:24:43,560 --> 00:24:44,544
AUDIENCE: Lit review.

519
00:24:44,544 --> 00:24:45,460
ELIZABETH NOLAN: Yeah.

520
00:24:45,460 --> 00:24:46,626
They went to the literature.

521
00:24:46,626 --> 00:24:48,606
And what did they find
in the literature?

522
00:24:52,378 --> 00:24:54,794
AUDIENCE: Someone had installed
a tRNA before or something

523
00:24:54,794 --> 00:24:57,142
and it didn't interfere
with the ribosome function.

524
00:24:57,142 --> 00:24:58,100
ELIZABETH NOLAN: Right.

525
00:24:58,100 --> 00:25:00,970
So they took
observations that were

526
00:25:00,970 --> 00:25:04,990
made from an independent group
for an independent project

527
00:25:04,990 --> 00:25:08,150
but the observations that were
useful to them in their design.

528
00:25:08,150 --> 00:25:12,460
So for some reason, this lab
stuck a tRNA onto the ribosome

529
00:25:12,460 --> 00:25:16,060
and saw the ribosome was still
active and functioning well.

530
00:25:16,060 --> 00:25:18,280
So the decision
was, why don't we

531
00:25:18,280 --> 00:25:21,890
use that to place the tag here?

532
00:25:21,890 --> 00:25:25,180
So what about their
choice of tag?

533
00:25:25,180 --> 00:25:25,945
What did they use?

534
00:25:30,457 --> 00:25:32,380
A big tag or a little tag?

535
00:25:37,528 --> 00:25:40,590
Any sense of that compared
to the size of, say, the 50S?

536
00:25:46,840 --> 00:25:48,190
Take a look at figure one.

537
00:26:07,152 --> 00:26:09,647
AUDIENCE: A small.

538
00:26:09,647 --> 00:26:11,870
ELIZABETH NOLAN: Yeah, right.

539
00:26:11,870 --> 00:26:16,280
So I'd say very small
compared to the size

540
00:26:16,280 --> 00:26:17,750
of the ribosome, right?

541
00:26:17,750 --> 00:26:20,360
So they decided to take
advantage of interaction

542
00:26:20,360 --> 00:26:27,050
between this MS2 coat protein
and the MS2 RNA recognition

543
00:26:27,050 --> 00:26:28,610
sequence, right?

544
00:26:28,610 --> 00:26:34,160
So that's one interaction
involved here,

545
00:26:34,160 --> 00:26:35,820
so ligand receptor interaction.

546
00:26:35,820 --> 00:26:39,770
So here, this is the depiction
from the paper showing

547
00:26:39,770 --> 00:26:48,075
where they incorporated this
MS2 stem loop into the 23S rRNA,

548
00:26:48,075 --> 00:26:49,100
here.

549
00:26:49,100 --> 00:26:57,075
And so what does that tag
need to bind, going back

550
00:26:57,075 --> 00:26:57,700
to the cartoon?

551
00:27:03,970 --> 00:27:06,290
And what is different
about this strategy

552
00:27:06,290 --> 00:27:09,680
from, say, what you've done with
nickel and TA chromatography

553
00:27:09,680 --> 00:27:12,050
and His6-tags?

554
00:27:12,050 --> 00:27:13,400
AUDIENCE: It has three things.

555
00:27:13,400 --> 00:27:14,970
ELIZABETH NOLAN:
Three things, yeah.

556
00:27:14,970 --> 00:27:15,470
Right?

557
00:27:15,470 --> 00:27:20,300
So we have three components,
two different interactions,

558
00:27:20,300 --> 00:27:24,170
say, between a ligand and
its binding partner, right?

559
00:27:24,170 --> 00:27:30,860
So here, we have the mutant
RNA of the ribosome where

560
00:27:30,860 --> 00:27:34,610
there's this MS2 stem loop, OK?

561
00:27:34,610 --> 00:27:39,680
That MS2 stem loop binds to
the MS2 coat protein, right?

562
00:27:39,680 --> 00:27:42,560
And then there needs to be
some way to pull this out.

563
00:27:42,560 --> 00:27:44,660
And what they chose
to do here was

564
00:27:44,660 --> 00:27:46,940
take advantage of a
second interaction and one

565
00:27:46,940 --> 00:27:50,330
that's commonly used in
chemistry and biology, which

566
00:27:50,330 --> 00:27:53,780
is looking at an interaction
between a protein called

567
00:27:53,780 --> 00:28:00,380
glutathione S-transferase
and glutathione, here, right?

568
00:28:00,380 --> 00:28:03,330
So effectively, they have
a solid support or a resin,

569
00:28:03,330 --> 00:28:05,690
so like the nickel NTA
column, but in this case,

570
00:28:05,690 --> 00:28:08,090
it's modified with GSH.

571
00:28:08,090 --> 00:28:11,420
They have to prepare
this fusion protein that

572
00:28:11,420 --> 00:28:15,350
is a fusion of GST
and MS2, and then they

573
00:28:15,350 --> 00:28:17,060
have the ribosome with the tag.

574
00:28:20,380 --> 00:28:22,960
So why might they have done
this with three components

575
00:28:22,960 --> 00:28:23,910
rather than two?

576
00:28:28,812 --> 00:28:35,660
AUDIENCE: Since GST/GSH, these
are pretty standard affinity

577
00:28:35,660 --> 00:28:39,395
tags, it was probably
easier to acquire GSH then

578
00:28:39,395 --> 00:28:40,624
to try to get MS2.

579
00:28:40,624 --> 00:28:41,540
ELIZABETH NOLAN: Yeah.

580
00:28:41,540 --> 00:28:46,420
That's a practical
analysis there.

581
00:28:46,420 --> 00:28:51,150
So could they have
made a resin with MS2?

582
00:28:51,150 --> 00:28:54,240
Maybe, right?

583
00:28:54,240 --> 00:29:04,190
So how did they go about doing
this affinity purification?

584
00:29:04,190 --> 00:29:06,650
What were the steps and why?

585
00:29:12,968 --> 00:29:19,660
So imagine they've done the
molecular biology required

586
00:29:19,660 --> 00:29:22,000
to express this tagged ribosome.

587
00:29:22,000 --> 00:29:26,020
They expressed the tagged
ribosome in E. coli.

588
00:29:26,020 --> 00:29:26,530
Then what?

589
00:29:26,530 --> 00:29:30,750
How are they going to get
this tagged ribosome out?

590
00:29:36,510 --> 00:29:39,870
AUDIENCE: They first purify
the crude ribosomes [INAUDIBLE]

591
00:29:39,870 --> 00:29:42,758
including the [INAUDIBLE]
and normal ones.

592
00:29:42,758 --> 00:29:47,520
And then they load these crude
samples through the column.

593
00:29:47,520 --> 00:29:48,620
ELIZABETH NOLAN: OK.

594
00:29:48,620 --> 00:29:51,470
So the crude sample went
through the column, right?

595
00:29:51,470 --> 00:29:54,050
What happened before that?

596
00:29:54,050 --> 00:29:57,440
So can you just put the crude
ribosome through the column,

597
00:29:57,440 --> 00:30:01,309
if your column is
the resin with GSH?

598
00:30:01,309 --> 00:30:03,600
AUDIENCE: You have to preload
it with a fusion protein.

599
00:30:03,600 --> 00:30:04,670
ELIZABETH NOLAN: Yes.

600
00:30:04,670 --> 00:30:07,334
So what did they preload
with the fusion protein?

601
00:30:10,652 --> 00:30:11,600
AUDIENCE: The column.

602
00:30:11,600 --> 00:30:12,475
ELIZABETH NOLAN: Yes.

603
00:30:12,475 --> 00:30:14,020
That's what they did, right?

604
00:30:14,020 --> 00:30:17,320
We can imagine just looking at
this without further details.

605
00:30:17,320 --> 00:30:21,160
There's two possibilities
with three components.

606
00:30:21,160 --> 00:30:25,120
So they could have, as stated
and as what they finally did,

607
00:30:25,120 --> 00:30:28,180
they could add this fusion
protein to the column, right?

608
00:30:28,180 --> 00:30:31,150
So the fusion protein binds GSH.

609
00:30:31,150 --> 00:30:36,640
And then you take your
crude lysate, crude material

610
00:30:36,640 --> 00:30:40,690
from the E. coli and run
that through the column

611
00:30:40,690 --> 00:30:42,880
to trap the ribosomes.

612
00:30:42,880 --> 00:30:44,350
What's the other possibility?

613
00:30:47,312 --> 00:30:49,020
They could have taken
this fusion protein

614
00:30:49,020 --> 00:30:51,660
and put it into
the crude mixture,

615
00:30:51,660 --> 00:30:52,920
and they talked about that.

616
00:30:52,920 --> 00:30:55,860
So what was one of the
complications with this fusion

617
00:30:55,860 --> 00:30:59,055
protein that led them to
incubate it with the column?

618
00:31:04,050 --> 00:31:06,370
Was this fusion
protein well-behaved?

619
00:31:26,010 --> 00:31:29,182
AUDIENCE: It forms
insoluble aggregate.

620
00:31:29,182 --> 00:31:30,390
ELIZABETH NOLAN: Yeah, right?

621
00:31:30,390 --> 00:31:32,480
It gave them some headaches.

622
00:31:32,480 --> 00:31:36,090
It aggregated.

623
00:31:36,090 --> 00:31:38,105
Is that something that
can commonly happen?

624
00:31:46,240 --> 00:31:50,320
So they likely tried both ways,
and they observed this problem

625
00:31:50,320 --> 00:31:54,790
with the fusion protein
having aggregation, right?

626
00:31:54,790 --> 00:31:57,670
And they avoided that
as being a complication

627
00:31:57,670 --> 00:32:00,430
to the purification by
incubating the column

628
00:32:00,430 --> 00:32:03,670
with that fusion protein first.

629
00:32:03,670 --> 00:32:09,100
So after they take
their crude sample

630
00:32:09,100 --> 00:32:12,400
and have that bind to
the resin in the column,

631
00:32:12,400 --> 00:32:18,200
how did they get the
ribosomes off the column?

632
00:32:18,200 --> 00:32:20,180
So what are the possibilities?

633
00:32:20,180 --> 00:32:22,420
And what did they
end up doing and why?

634
00:32:28,144 --> 00:32:32,190
AUDIENCE: So you could just
use an excessive of free MS2

635
00:32:32,190 --> 00:32:34,630
ligands to cause the
ribosomes to disassociate

636
00:32:34,630 --> 00:32:35,572
from the column.

637
00:32:35,572 --> 00:32:37,927
Or you could use an
excess of the GST

638
00:32:37,927 --> 00:32:40,784
to cause that complex to
dissociate from the column.

639
00:32:40,784 --> 00:32:41,700
ELIZABETH NOLAN: Yeah.

640
00:32:41,700 --> 00:32:46,590
So thinking about the
latter possibility--

641
00:32:46,590 --> 00:32:48,660
so what Rebecca has
done is identify

642
00:32:48,660 --> 00:32:53,340
the two different ligand
receptor interactions, right?

643
00:32:53,340 --> 00:32:57,450
We could disrupt this one
between MS2 fusion coat

644
00:32:57,450 --> 00:33:01,710
protein and the ribosome
or between GSH and GST.

645
00:33:01,710 --> 00:33:06,690
So in terms of this one
here, does it make more sense

646
00:33:06,690 --> 00:33:12,510
to elute with excess protein
or excess glutathione, which

647
00:33:12,510 --> 00:33:14,100
is effectively a tripeptide?

648
00:33:17,514 --> 00:33:18,430
AUDIENCE: Glutathione.

649
00:33:18,430 --> 00:33:19,638
ELIZABETH NOLAN: Yeah, right?

650
00:33:19,638 --> 00:33:24,790
So just that much easier to
come by a lot of glutathione

651
00:33:24,790 --> 00:33:26,510
than a lot of GSH--

652
00:33:26,510 --> 00:33:28,150
or sorry, GST.

653
00:33:28,150 --> 00:33:29,750
So which one did they choose?

654
00:33:34,115 --> 00:33:36,690
How did they elute the
ribosome off the column?

655
00:33:42,460 --> 00:33:45,700
How long did you each spend on
the paper before coming here?

656
00:33:48,538 --> 00:33:49,960
AUDIENCE: GSH.

657
00:33:49,960 --> 00:33:52,120
ELIZABETH NOLAN:
Yeah, they used GSH.

658
00:33:52,120 --> 00:33:55,090
So they eluted with excess GSH.

659
00:33:55,090 --> 00:34:00,100
So what does that mean in
terms of the purified ribosome?

660
00:34:00,100 --> 00:34:02,110
Is it just the ribosome
with the stem loop?

661
00:34:02,110 --> 00:34:03,060
AUDIENCE: They still
have the fusion protein.

662
00:34:03,060 --> 00:34:04,018
ELIZABETH NOLAN: Right.

663
00:34:04,018 --> 00:34:06,460
We still have the
fusion protein on.

664
00:34:06,460 --> 00:34:10,540
So if you were making this
decision at the bench,

665
00:34:10,540 --> 00:34:14,320
you have two different
interactions to consider,

666
00:34:14,320 --> 00:34:17,266
what would you choose and why?

667
00:34:17,266 --> 00:34:19,030
Why and I guess,
really, why did they

668
00:34:19,030 --> 00:34:22,530
choose to disrupt the
interaction between GSH

669
00:34:22,530 --> 00:34:23,030
and GST?

670
00:34:29,740 --> 00:34:36,600
AUDIENCE: Because it's difficult
to have enough MS2 than

671
00:34:36,600 --> 00:34:38,042
to purify with glutathione.

672
00:34:38,042 --> 00:34:39,250
ELIZABETH NOLAN: Here, right?

673
00:34:39,250 --> 00:34:44,080
Because if you were going to,
say, elute with excess of MS2

674
00:34:44,080 --> 00:34:47,620
stem loop, where
would that come from?

675
00:34:47,620 --> 00:34:50,100
Or excess MS2 protein, right?

676
00:34:50,100 --> 00:34:51,935
AUDIENCE: Also,
it's actually MS2.

677
00:34:51,935 --> 00:34:52,810
ELIZABETH NOLAN: Yes.

678
00:34:52,810 --> 00:34:55,630
It's not very
practical here, right?

679
00:34:55,630 --> 00:34:57,760
At the end of the
day, it would be best

680
00:34:57,760 --> 00:35:00,880
to have the ribosome without
this fusion protein attached,

681
00:35:00,880 --> 00:35:04,030
but disrupting this interaction
isn't very practical.

682
00:35:04,030 --> 00:35:06,610
So it would be quite
expensive either way

683
00:35:06,610 --> 00:35:08,787
if you were making a lot
of some sort of stem loop.

684
00:35:08,787 --> 00:35:10,870
And then how would you
even know you have the stem

685
00:35:10,870 --> 00:35:14,750
loop or the protein here?

686
00:35:14,750 --> 00:35:15,850
OK.

687
00:35:15,850 --> 00:35:18,790
So I'm just curious,
for those of you

688
00:35:18,790 --> 00:35:21,670
who have done like nickel
NTA chromatography,

689
00:35:21,670 --> 00:35:26,320
do you have a sense of the
affinity of the His-tag protein

690
00:35:26,320 --> 00:35:29,030
for the resin?

691
00:35:29,030 --> 00:35:32,425
So what happens as this tied
protein goes down the column?

692
00:35:40,165 --> 00:35:40,670
All right.

693
00:35:40,670 --> 00:35:58,286
So you have some column with
your resin plus His6 protein.

694
00:36:13,036 --> 00:36:17,482
So was it a strong
or weak interaction?

695
00:36:17,482 --> 00:36:18,190
AUDIENCE: Strong.

696
00:36:18,190 --> 00:36:19,110
AUDIENCE: Strong.

697
00:36:19,110 --> 00:36:21,526
ELIZABETH NOLAN: How
would you define strong?

698
00:36:21,526 --> 00:36:23,896
Or why do you say it's strong?

699
00:36:23,896 --> 00:36:26,429
AUDIENCE: The chelation.

700
00:36:26,429 --> 00:36:28,720
ELIZABETH NOLAN: Well, you're
forming a complex, right?

701
00:36:28,720 --> 00:36:32,330
You're forming-- the His-tag
is binding the nickel NTA.

702
00:36:32,330 --> 00:36:36,470
AUDIENCE: The Kd is
probably [INAUDIBLE]..

703
00:36:36,470 --> 00:36:37,866
ELIZABETH NOLAN: Is it?

704
00:36:37,866 --> 00:36:39,285
AUDIENCE: I don't know.

705
00:36:39,285 --> 00:36:41,028
AUDIENCE: It is
a dynamic process

706
00:36:41,028 --> 00:36:43,069
where they're releasing
and binding and releasing

707
00:36:43,069 --> 00:36:44,252
and binding again.

708
00:36:46,850 --> 00:36:47,900
ELIZABETH NOLAN: Yes.

709
00:36:47,900 --> 00:36:49,920
So there's an
equilibrium, right?

710
00:36:49,920 --> 00:36:53,080
And we talk about
binding to the column

711
00:36:53,080 --> 00:36:56,720
but how tightly is this
tagged protein binding?

712
00:36:56,720 --> 00:36:58,850
And is it just binding
there and getting stuck?

713
00:36:58,850 --> 00:37:02,360
I mean, it needs to stay
in your column, right?

714
00:37:02,360 --> 00:37:05,190
In this case, it's
not very strong.

715
00:37:05,190 --> 00:37:07,580
So if you look at
reported Kd's for, say,

716
00:37:07,580 --> 00:37:09,890
His-tags to nickel NTA,
they're on the order

717
00:37:09,890 --> 00:37:12,470
of one to 10 micromolar.

718
00:37:12,470 --> 00:37:16,175
So orders of magnitude
lower affinity

719
00:37:16,175 --> 00:37:17,900
than what you just suggested.

720
00:37:17,900 --> 00:37:21,970
So why does the column work?

721
00:37:21,970 --> 00:37:24,419
AUDIENCE: Because everything
else binds worse than that.

722
00:37:24,419 --> 00:37:25,960
ELIZABETH NOLAN:
Well, you hope that.

723
00:37:25,960 --> 00:37:26,459
You hope.

724
00:37:26,459 --> 00:37:28,312
I mean, sure.

725
00:37:28,312 --> 00:37:30,520
I mean, if you know that
histamine-- a protein that's

726
00:37:30,520 --> 00:37:34,720
histamine-rich it's going to
stick, but why does it work?

727
00:37:37,750 --> 00:37:41,860
So is it surprising that
a micromolar affinity

728
00:37:41,860 --> 00:37:46,300
can allow this to be
trapped on the column?

729
00:37:46,300 --> 00:37:49,730
AUDIENCE: Is it because you
have six histamines tied down

730
00:37:49,730 --> 00:37:56,080
[INAUDIBLE]

731
00:37:56,080 --> 00:37:57,080
ELIZABETH NOLAN: Pardon?

732
00:38:00,510 --> 00:38:01,030
OK.

733
00:38:01,030 --> 00:38:04,580
It's not the amount
of histamines.

734
00:38:04,580 --> 00:38:06,687
What is in your column?

735
00:38:06,687 --> 00:38:08,520
AUDIENCE: You've got a
lot of binding sites.

736
00:38:08,520 --> 00:38:08,850
ELIZABETH NOLAN: Yeah.

737
00:38:08,850 --> 00:38:09,430
Right.

738
00:38:09,430 --> 00:38:13,220
There's a lot of binding
sites in the column.

739
00:38:13,220 --> 00:38:15,410
So you're going to have,
as Rebecca said, dynamic.

740
00:38:15,410 --> 00:38:17,212
This is coming on
and off the column,

741
00:38:17,212 --> 00:38:18,670
but there's a lot
of binding sites.

742
00:38:18,670 --> 00:38:21,520
So if it comes off, it
can go back on there.

743
00:38:25,822 --> 00:38:29,215
So what about the GST and GSH?

744
00:38:35,450 --> 00:38:37,925
AUDIENCE: I know it's one of
the strongest attractions.

745
00:38:37,925 --> 00:38:38,841
ELIZABETH NOLAN: Yeah.

746
00:38:38,841 --> 00:38:40,860
This is much stronger
than nickel NTA

747
00:38:40,860 --> 00:38:42,600
and the His-tag protein.

748
00:38:42,600 --> 00:38:46,766
So orders of magnitude higher
affinity here for that.

749
00:38:46,766 --> 00:38:47,536
OK.

750
00:38:47,536 --> 00:38:49,410
But it's something to
think about when you're

751
00:38:49,410 --> 00:38:54,384
choosing an affinity
purification method there

752
00:38:54,384 --> 00:38:55,800
and to think about
what's actually

753
00:38:55,800 --> 00:39:02,330
happening on this column
and dynamic process.

754
00:39:02,330 --> 00:39:09,780
So jumping ahead a little bit,
they did their experiments

755
00:39:09,780 --> 00:39:13,622
first just tagging the
wild-type ribosome, OK?

756
00:39:13,622 --> 00:39:15,330
And that's very
important because they're

757
00:39:15,330 --> 00:39:17,790
trying to make a new
purification method,

758
00:39:17,790 --> 00:39:19,980
and the first thing
that needs to be asked

759
00:39:19,980 --> 00:39:23,940
is does the wild-type
ribosome plus the tag

760
00:39:23,940 --> 00:39:28,470
behave the same or differently
from the wild-type ribosome

761
00:39:28,470 --> 00:39:30,330
without the tag, OK?

762
00:39:30,330 --> 00:39:32,370
And you want to know that
because if the tag is

763
00:39:32,370 --> 00:39:34,702
causing a problem, maybe
it's not a good design.

764
00:39:34,702 --> 00:39:36,660
And you don't want to go
forward making mutants

765
00:39:36,660 --> 00:39:41,100
with that kind of
modification, OK?

766
00:39:41,100 --> 00:39:49,500
So what do they need to do after
doing this purification, right?

767
00:39:49,500 --> 00:39:53,910
One is just analyzing the
purity of the material

768
00:39:53,910 --> 00:39:55,440
that they've come up with.

769
00:39:55,440 --> 00:39:57,390
And then the other
things they did

770
00:39:57,390 --> 00:39:59,940
was look at the subunit
integrity, right?

771
00:39:59,940 --> 00:40:03,000
And so something to keep in
mind is that the ribosome

772
00:40:03,000 --> 00:40:04,050
has two subunits.

773
00:40:04,050 --> 00:40:07,110
It has many ribosomal proteins.

774
00:40:07,110 --> 00:40:10,770
Does putting the tag on
only one subunit work well?

775
00:40:10,770 --> 00:40:14,490
And then, of course, they need
to think about the activity.

776
00:40:14,490 --> 00:40:16,770
And so they presented a
number of different assays

777
00:40:16,770 --> 00:40:21,060
in this paper ranging
from looking at kinetics

778
00:40:21,060 --> 00:40:23,730
of peptide bond
formation to looking

779
00:40:23,730 --> 00:40:29,100
at kinetic studies of release
with one of the release factors

780
00:40:29,100 --> 00:40:30,280
here.

781
00:40:30,280 --> 00:40:34,590
So let's think about
the purity analysis.

782
00:40:34,590 --> 00:40:38,310
And what we're going to focus
on is there chromatogram, right?

783
00:40:38,310 --> 00:40:43,680
So as we know, they took
their column of GSH.

784
00:40:43,680 --> 00:40:47,130
They first loaded that column
with the GST/MS2 fusion

785
00:40:47,130 --> 00:40:52,080
protein, and then they
added their crude sample

786
00:40:52,080 --> 00:40:54,580
and eluted with GSH, right?

787
00:40:54,580 --> 00:40:58,410
And so the data they present for
the chromatogram for monitoring

788
00:40:58,410 --> 00:41:02,370
fractions of that column
is shown here, OK?

789
00:41:02,370 --> 00:41:04,980
So what does this tell us?

790
00:41:04,980 --> 00:41:08,237
What do we see in
this chromatogram?

791
00:41:13,704 --> 00:41:15,636
So what are they monitoring?

792
00:41:21,980 --> 00:41:25,270
So first you want to
read your axes, right?

793
00:41:25,270 --> 00:41:27,602
So what are they monitoring.

794
00:41:27,602 --> 00:41:28,974
AUDIENCE: A260.

795
00:41:28,974 --> 00:41:30,640
ELIZABETH NOLAN: Yes,
that's the y-axis.

796
00:41:30,640 --> 00:41:32,290
And why A260?

797
00:41:32,290 --> 00:41:34,654
Going back to 20 minutes ago.

798
00:41:34,654 --> 00:41:35,410
AUDIENCE: DNA.

799
00:41:35,410 --> 00:41:36,910
ELIZABETH NOLAN:
Nucleotides, right?

800
00:41:36,910 --> 00:41:38,340
Do we want to monitor DNA here?

801
00:41:41,260 --> 00:41:42,730
It will work for DNA.

802
00:41:42,730 --> 00:41:43,614
AUDIENCE: Oh, RNA

803
00:41:43,614 --> 00:41:44,530
ELIZABETH NOLAN: Yeah.

804
00:41:44,530 --> 00:41:48,850
So we have the 23S, 16S rRNA.

805
00:41:48,850 --> 00:41:51,430
So we're looking at
A260, which makes sense.

806
00:41:51,430 --> 00:41:54,600
We're trying to
purify the ribosome.

807
00:41:54,600 --> 00:41:56,590
Volume, what is this volume?

808
00:42:01,147 --> 00:42:02,356
AUDIENCE: The elution volume.

809
00:42:02,356 --> 00:42:03,313
ELIZABETH NOLAN: Right.

810
00:42:03,313 --> 00:42:04,970
The volume eluted
from the column.

811
00:42:04,970 --> 00:42:08,540
So looking at this
trace, what do we see?

812
00:42:12,033 --> 00:42:15,526
AUDIENCE: There's
a peak [INAUDIBLE]

813
00:42:15,526 --> 00:42:18,530
maybe there's some
[INAUDIBLE] introduced later.

814
00:42:18,530 --> 00:42:19,600
ELIZABETH NOLAN: OK.

815
00:42:19,600 --> 00:42:22,610
So you've mixed what you
see with an interpretation.

816
00:42:22,610 --> 00:42:25,932
So let's just stick right now
to what do we see in the trace?

817
00:42:25,932 --> 00:42:27,640
And then we're going
to think about where

818
00:42:27,640 --> 00:42:28,810
these things come from.

819
00:42:28,810 --> 00:42:31,630
And that's just
something important,

820
00:42:31,630 --> 00:42:33,790
I think, with the problems
we give in this course.

821
00:42:33,790 --> 00:42:37,050
First, you want to ask just
what does the data say?

822
00:42:37,050 --> 00:42:39,280
And then, how do we
interpret this data

823
00:42:39,280 --> 00:42:42,384
based on our knowledge
of a system here, OK?

824
00:42:42,384 --> 00:42:44,800
So do you see what I mean, how
you mixed what you see here

825
00:42:44,800 --> 00:42:47,050
and a potential interpretation?

826
00:42:47,050 --> 00:42:49,328
So just what do we see?

827
00:42:49,328 --> 00:42:50,462
AUDIENCE: Two peaks.

828
00:42:50,462 --> 00:42:51,670
ELIZABETH NOLAN: So there's--

829
00:42:51,670 --> 00:42:52,169
Yeah.

830
00:42:52,169 --> 00:42:53,432
Rebecca?

831
00:42:53,432 --> 00:42:56,869
AUDIENCE: Just the
large broad peak

832
00:42:56,869 --> 00:43:02,270
that results immediately, and
then a smaller separate peak.

833
00:43:02,270 --> 00:43:04,640
ELIZABETH NOLAN: So that's
a very nice description.

834
00:43:04,640 --> 00:43:09,680
We see a broad peak with
high A260 absorbance,

835
00:43:09,680 --> 00:43:11,960
then it elutes immediately.

836
00:43:11,960 --> 00:43:15,890
And then later, there's a
peak just after 30 mils that's

837
00:43:15,890 --> 00:43:17,150
sharper, right?

838
00:43:17,150 --> 00:43:18,725
So what are these peaks?

839
00:43:23,405 --> 00:43:24,790
What came off here?

840
00:43:31,552 --> 00:43:33,001
AUDIENCE: Everything else.

841
00:43:33,001 --> 00:43:33,970
ELIZABETH NOLAN: Yeah.

842
00:43:33,970 --> 00:43:35,400
So what's everything else?

843
00:43:40,190 --> 00:43:44,064
AUDIENCE: DNA, [INAUDIBLE].

844
00:43:44,064 --> 00:43:44,980
ELIZABETH NOLAN: Yeah.

845
00:43:44,980 --> 00:43:47,320
So things that didn't
stick to the column, right?

846
00:43:47,320 --> 00:43:48,390
They got washed out.

847
00:43:48,390 --> 00:43:51,700
So maybe the native
ribosomes, right?

848
00:43:51,700 --> 00:43:54,700
Maybe there's DNA
in there, tRNAs.

849
00:43:54,700 --> 00:43:57,940
Could there be EFTU
with tRNA bound, right?

850
00:43:57,940 --> 00:44:03,100
And there are things
we're not seeing, right?

851
00:44:03,100 --> 00:44:06,250
So what do we think
about the second peak?

852
00:44:11,840 --> 00:44:13,230
AUDIENCE: It's much less broad.

853
00:44:13,230 --> 00:44:15,000
It's pretty sharp,
so that will tell you

854
00:44:15,000 --> 00:44:18,835
that it's likely
only one thing that

855
00:44:18,835 --> 00:44:20,621
has bound to the column a lot.

856
00:44:20,621 --> 00:44:22,245
So that would probably
be your His-tag.

857
00:44:22,245 --> 00:44:23,924
ELIZABETH NOLAN: Is
this a His-tag here?

858
00:44:23,924 --> 00:44:26,090
I know we're going back and
forth because you're all

859
00:44:26,090 --> 00:44:27,256
more familiar with His-tags.

860
00:44:27,256 --> 00:44:28,912
AUDIENCE: Your affinity tag.

861
00:44:28,912 --> 00:44:29,870
ELIZABETH NOLAN: Right.

862
00:44:29,870 --> 00:44:32,990
So this is likely what
was tagged, right?

863
00:44:32,990 --> 00:44:36,860
And with GSH
elution, we disrupted

864
00:44:36,860 --> 00:44:38,600
the binding
interaction with GST,

865
00:44:38,600 --> 00:44:40,190
and it came off the column.

866
00:44:40,190 --> 00:44:44,240
Do we know that
it's only one thing?

867
00:44:44,240 --> 00:44:45,560
No.

868
00:44:45,560 --> 00:44:47,570
At this stage, we don't, right?

869
00:44:47,570 --> 00:44:50,515
So what are possibilities?

870
00:44:50,515 --> 00:44:52,850
What could this be?

871
00:44:52,850 --> 00:44:54,550
So we have the tag on the 50S.

872
00:45:04,142 --> 00:45:06,100
Because the only-- I
mean, it's coming off here

873
00:45:06,100 --> 00:45:09,790
just based on the amount of
GSH required to push it off

874
00:45:09,790 --> 00:45:10,856
the column there.

875
00:45:10,856 --> 00:45:13,231
AUDIENCE: It could be a mixture
of intact ribosome, which

876
00:45:13,231 --> 00:45:14,832
is the cell that gets tagged.

877
00:45:14,832 --> 00:45:15,790
ELIZABETH NOLAN: Right.

878
00:45:15,790 --> 00:45:16,290
Yeah.

879
00:45:16,290 --> 00:45:19,090
So we don't know right now in
terms of the whole composition

880
00:45:19,090 --> 00:45:20,740
of this peak, right?

881
00:45:20,740 --> 00:45:24,460
It could be intact 70S
because 30S came down.

882
00:45:24,460 --> 00:45:26,970
It could be 50S alone.

883
00:45:26,970 --> 00:45:28,660
And there's always
the possibility

884
00:45:28,660 --> 00:45:31,120
of some other contaminant
that just for whatever reason

885
00:45:31,120 --> 00:45:33,830
came off the column then there.

886
00:45:33,830 --> 00:45:34,880
OK.

887
00:45:34,880 --> 00:45:37,000
Would you be excited
by this chromatogram

888
00:45:37,000 --> 00:45:39,580
if you were the person at
the bench doing this work?

889
00:45:44,283 --> 00:45:44,783
Yeah.

890
00:45:44,783 --> 00:45:47,030
You'd be super excited, right?

891
00:45:47,030 --> 00:45:50,320
So it looks quite good.

892
00:45:50,320 --> 00:45:53,090
So of course, there needs to
be some more analyses done.

893
00:45:53,090 --> 00:45:55,820
And one analysis we're not
going to go into in detail

894
00:45:55,820 --> 00:45:59,630
but they needed to ask, is
this really all tagged ribosome

895
00:45:59,630 --> 00:46:02,180
or is there also some
contamination of wild-type?

896
00:46:02,180 --> 00:46:06,530
And so they designed some
analysis using a technique

897
00:46:06,530 --> 00:46:09,380
called primary extension
to look at that there.

898
00:46:09,380 --> 00:46:12,080
And what they saw is
that they primarily

899
00:46:12,080 --> 00:46:15,480
had the tagged ribosome,
which was good news.

900
00:46:15,480 --> 00:46:17,780
So getting at the question
in terms of what's actually

901
00:46:17,780 --> 00:46:21,050
in this peak, they
looked at effectively,

902
00:46:21,050 --> 00:46:24,095
say, subunit integrity.

903
00:46:24,095 --> 00:46:25,880
And how did they do this?

904
00:46:25,880 --> 00:46:28,492
They used centrifugation.

905
00:46:28,492 --> 00:46:31,640
And does anyone recall what type
of centrifugation they used?

906
00:46:36,262 --> 00:46:37,720
AUDIENCE: Sucrose
density gradient.

907
00:46:37,720 --> 00:46:38,595
ELIZABETH NOLAN: Yes.

908
00:46:38,595 --> 00:46:41,120
So they used a sucrose
gradient, right?

909
00:46:41,120 --> 00:46:43,696
And how does that let
you do separation?

910
00:46:43,696 --> 00:46:44,602
AUDIENCE: By density.

911
00:46:44,602 --> 00:46:46,060
ELIZABETH NOLAN:
Right, by density.

912
00:46:46,060 --> 00:46:50,090
And we have the different
subunits of different sizes,

913
00:46:50,090 --> 00:46:52,060
different density there, right?

914
00:46:52,060 --> 00:46:56,780
So this is the data they show.

915
00:46:56,780 --> 00:47:00,490
And so, again, we want to
look at these data and ask

916
00:47:00,490 --> 00:47:03,520
what do we see and what
does that tell us, right?

917
00:47:03,520 --> 00:47:07,120
So these are the results
from the sucrose gradient

918
00:47:07,120 --> 00:47:08,470
centrifugation.

919
00:47:08,470 --> 00:47:12,920
And they looked at untied
wild-type ribosomes,

920
00:47:12,920 --> 00:47:16,810
so isolated 70S and
then the tied ribosome.

921
00:47:16,810 --> 00:47:20,680
So what was the key part of
the sample preparation here?

922
00:47:23,200 --> 00:47:25,090
How did they prepare
these samples

923
00:47:25,090 --> 00:47:27,820
to be able to look at the
subunits individually?

924
00:47:33,199 --> 00:47:34,990
Because the question
they're getting at is,

925
00:47:34,990 --> 00:47:39,520
what is the ratio of the 50S
to the 30S in the sample that

926
00:47:39,520 --> 00:47:42,912
was purified from the column?

927
00:47:42,912 --> 00:47:47,374
AUDIENCE: They dialyzed
using magnesium buffer.

928
00:47:47,374 --> 00:47:48,290
ELIZABETH NOLAN: Yeah.

929
00:47:48,290 --> 00:47:50,690
So what was it about the
magnesium in the buffer?

930
00:47:54,022 --> 00:47:55,926
AUDIENCE: It causes
disassociation

931
00:47:55,926 --> 00:47:57,840
between the 30S and the 50S.

932
00:47:57,840 --> 00:47:58,862
ELIZABETH NOLAN: So why?

933
00:47:58,862 --> 00:48:00,570
Maybe you said it,
and I didn't hear you.

934
00:48:00,570 --> 00:48:03,969
What was it about the
buffer that allowed this?

935
00:48:03,969 --> 00:48:07,079
AUDIENCE: Is it the
positive charge?

936
00:48:07,079 --> 00:48:09,495
ELIZABETH NOLAN: Well, it is
the magnesium, but was it low

937
00:48:09,495 --> 00:48:10,560
or high magnesium buffer?

938
00:48:10,560 --> 00:48:11,310
AUDIENCE: Oh, low.

939
00:48:11,310 --> 00:48:12,630
ELIZABETH NOLAN: Low, Right?

940
00:48:12,630 --> 00:48:15,960
So we learned that magnesium
is important for interactions

941
00:48:15,960 --> 00:48:17,530
between these subunits, right?

942
00:48:17,530 --> 00:48:20,430
And you've seen some of
the experimental details

943
00:48:20,430 --> 00:48:22,800
in the paper for
recitation two and three,

944
00:48:22,800 --> 00:48:25,530
they used different
concentrations of magnesium.

945
00:48:25,530 --> 00:48:28,140
In this case, they want to
separate the two subunits.

946
00:48:28,140 --> 00:48:32,340
So they basically used
a low magnesium buffer

947
00:48:32,340 --> 00:48:36,570
to allow them to dissociate.

948
00:48:36,570 --> 00:48:39,720
So in these data, what
are we looking at?

949
00:48:39,720 --> 00:48:41,670
The axes aren't shown,
but what are they?

950
00:48:46,320 --> 00:48:47,715
AUDIENCE: I have a question.

951
00:48:47,715 --> 00:48:50,046
Why not just a
no-magnesium buffer?

952
00:48:55,730 --> 00:48:58,950
ELIZABETH NOLAN: Could
that be bad for the sample?

953
00:48:58,950 --> 00:49:01,629
How low is the
magnesium in the buffer?

954
00:49:01,629 --> 00:49:02,670
AUDIENCE: One millimolar.

955
00:49:02,670 --> 00:49:04,260
ELIZABETH NOLAN: One millimolar.

956
00:49:04,260 --> 00:49:08,060
So where else might
the magnesium go?

957
00:49:08,060 --> 00:49:10,353
Is it only important
for this interaction?

958
00:49:20,644 --> 00:49:22,185
AUDIENCE: I mean I
guess it's useful.

959
00:49:22,185 --> 00:49:23,976
It can be used in a
lot of different places

960
00:49:23,976 --> 00:49:25,197
actually in the cell.

961
00:49:25,197 --> 00:49:26,030
ELIZABETH NOLAN: OK.

962
00:49:26,030 --> 00:49:27,540
But we don't have the cell here.

963
00:49:27,540 --> 00:49:29,174
We have the purified ribosome.

964
00:49:29,174 --> 00:49:30,590
AUDIENCE: Is it
possible that it's

965
00:49:30,590 --> 00:49:32,820
holding together the
actual conformation

966
00:49:32,820 --> 00:49:35,032
of the 50S and the 30S?

967
00:49:35,032 --> 00:49:36,240
ELIZABETH NOLAN: Yeah, right.

968
00:49:36,240 --> 00:49:37,375
I mean, that may--

969
00:49:37,375 --> 00:49:39,000
I actually don't know
what would happen

970
00:49:39,000 --> 00:49:41,460
if this goes into a
no-magnesium buffer, right?

971
00:49:41,460 --> 00:49:44,490
But I think there's about
a dozen contacts where

972
00:49:44,490 --> 00:49:46,614
magnesium is used
between the two subunits.

973
00:49:46,614 --> 00:49:48,030
And you can imagine
there's plenty

974
00:49:48,030 --> 00:49:50,190
of interactions of
magnesium or cations

975
00:49:50,190 --> 00:49:52,121
in other places of each subunit.

976
00:49:52,121 --> 00:49:54,620
Joanne, do you happen to know
what happens if the ribosome--

977
00:49:54,620 --> 00:49:56,335
I think you get a
big unfolded mess.

978
00:49:56,335 --> 00:49:58,793
JOANNE STUBBE: It would be hard
to get rid of the magnesium

979
00:49:58,793 --> 00:50:01,477
because it has key binding
sites along the place

980
00:50:01,477 --> 00:50:05,330
where it comes off
and goes back on.

981
00:50:05,330 --> 00:50:06,630
ELIZABETH NOLAN: Yeah.

982
00:50:06,630 --> 00:50:08,100
So what are the axes?

983
00:50:08,100 --> 00:50:10,100
If we were to have them,
what are we looking at?

984
00:50:13,022 --> 00:50:15,424
AUDIENCE: I think the gradient.

985
00:50:15,424 --> 00:50:16,340
ELIZABETH NOLAN: Yeah.

986
00:50:16,340 --> 00:50:16,839
Right.

987
00:50:16,839 --> 00:50:19,550
So we have basically,
yeah, the percent sucrose,

988
00:50:19,550 --> 00:50:22,730
say, the gradient--
or the density, right?

989
00:50:22,730 --> 00:50:25,670
And how are we
seeing these peaks?

990
00:50:35,800 --> 00:50:40,700
AUDIENCE: We see two
peaks and the upper graph

991
00:50:40,700 --> 00:50:46,250
we see the peaks are
more similar in size.

992
00:50:46,250 --> 00:50:51,150
And then in the second part we
see-- well, we see two peaks

993
00:50:51,150 --> 00:50:53,600
but one peak has a shift over.

994
00:50:53,600 --> 00:50:55,640
ELIZABETH NOLAN: Right.

995
00:50:55,640 --> 00:50:57,470
So just backing up.

996
00:50:57,470 --> 00:50:59,725
That's all certainly the case.

997
00:50:59,725 --> 00:51:02,060
How are we detecting
these peaks?

998
00:51:02,060 --> 00:51:03,140
What's the readout?

999
00:51:03,140 --> 00:51:07,200
So we have some
sucrose gradient here.

1000
00:51:07,200 --> 00:51:09,057
How do we see the peaks?

1001
00:51:09,057 --> 00:51:10,370
AUDIENCE: PUB.

1002
00:51:10,370 --> 00:51:12,400
ELIZABETH NOLAN: Well,
what, more specifically?

1003
00:51:12,400 --> 00:51:13,710
AUDIENCE: Oh, A280.

1004
00:51:13,710 --> 00:51:14,626
ELIZABETH NOLAN: Yeah.

1005
00:51:14,626 --> 00:51:17,660
So here, they're using the
absorbance at 280, right?

1006
00:51:17,660 --> 00:51:23,611
So a little different than the
chromatogram, but that's OK,

1007
00:51:23,611 --> 00:51:24,110
right?

1008
00:51:24,110 --> 00:51:28,025
There's 280 absorbance here and
maybe their instrument for this

1009
00:51:28,025 --> 00:51:29,820
didn't allow 260.

1010
00:51:29,820 --> 00:51:36,660
So as we just heard, if we
look at the untagged ribosome,

1011
00:51:36,660 --> 00:51:40,380
we see that there's two peaks,
and they're nicely labeled.

1012
00:51:40,380 --> 00:51:43,340
It's nicely labeled
with the 30S here

1013
00:51:43,340 --> 00:51:49,021
in terms of percent sucrose
in the gradient and 50S here,

1014
00:51:49,021 --> 00:51:49,520
right?

1015
00:51:49,520 --> 00:51:53,360
And so we see kind of
in our standard control,

1016
00:51:53,360 --> 00:51:56,180
the peak for the 30S is
smaller than the 50S,

1017
00:51:56,180 --> 00:51:57,710
and this is where
they're placed.

1018
00:51:57,710 --> 00:52:02,120
And so what we want to do is
compare this data to the data

1019
00:52:02,120 --> 00:52:04,020
here for the tagged ribosome.

1020
00:52:04,020 --> 00:52:04,520
OK.

1021
00:52:04,520 --> 00:52:07,670
So what are the two
major observations,

1022
00:52:07,670 --> 00:52:09,237
two major differences?

1023
00:52:12,646 --> 00:52:14,594
Rebecca.

1024
00:52:14,594 --> 00:52:18,003
AUDIENCE: The second one's
enriched for the proteasome,

1025
00:52:18,003 --> 00:52:20,940
there's relatively higher
concentration of 50S.

1026
00:52:20,940 --> 00:52:22,230
ELIZABETH NOLAN: OK and--

1027
00:52:22,230 --> 00:52:24,376
AUDIENCE: And it's [INAUDIBLE].

1028
00:52:24,376 --> 00:52:25,250
ELIZABETH NOLAN: Yes.

1029
00:52:25,250 --> 00:52:27,480
So or another way to
say that, maybe, it's

1030
00:52:27,480 --> 00:52:30,570
depleted in the 30S, right?

1031
00:52:30,570 --> 00:52:33,870
But I think you're coming across
the same observation, right?

1032
00:52:33,870 --> 00:52:39,559
So first we see there is a
peak corresponding to the 30S

1033
00:52:39,559 --> 00:52:41,100
and then there's
this peak here which

1034
00:52:41,100 --> 00:52:42,780
is shifted relative
to what we saw

1035
00:52:42,780 --> 00:52:45,240
for the 50S and the
untagged ribosome.

1036
00:52:45,240 --> 00:52:47,440
And the peak heights
or peak areas,

1037
00:52:47,440 --> 00:52:49,880
however you want to
analyze it, that ratio

1038
00:52:49,880 --> 00:52:53,170
is very different than
what we see up here.

1039
00:52:53,170 --> 00:52:56,990
So why is this shifted
and why is there more 50S?

1040
00:53:00,670 --> 00:53:02,090
AUDIENCE: Can I ask a question?

1041
00:53:02,090 --> 00:53:05,990
So can you definitively say
that it's enriched or depleted

1042
00:53:05,990 --> 00:53:08,050
in 30S?

1043
00:53:08,050 --> 00:53:10,690
I don't know a lot about
biochemistry in general,

1044
00:53:10,690 --> 00:53:17,010
but can you say that possibly
the multiple histamine--

1045
00:53:17,010 --> 00:53:18,686
oh, it's not histamine.

1046
00:53:21,711 --> 00:53:24,470
AUDIENCE: Is it just tagged
because of the shift.

1047
00:53:24,470 --> 00:53:26,750
I mean, it seems like
such a small derivation.

1048
00:53:26,750 --> 00:53:27,583
ELIZABETH NOLAN: OK.

1049
00:53:27,583 --> 00:53:30,200
So what else is
there in the sample?

1050
00:53:30,200 --> 00:53:33,400
So we have that stem loop tag,
but based on how they eluted

1051
00:53:33,400 --> 00:53:35,410
this, what else is there?

1052
00:53:35,410 --> 00:53:36,310
AUDIENCE: The fusion.

1053
00:53:36,310 --> 00:53:38,250
ELIZABETH NOLAN: The
fusion protein, right?

1054
00:53:38,250 --> 00:53:40,000
So then the question
is-- and this is just

1055
00:53:40,000 --> 00:53:42,416
getting to the point of needing
to look at all the details

1056
00:53:42,416 --> 00:53:45,070
in terms of what's done in
someone's experiment to try

1057
00:53:45,070 --> 00:53:47,470
to figure out what's happening.

1058
00:53:47,470 --> 00:53:51,730
Is the MS2 coat protein alone
enough to cause a shift?

1059
00:53:54,250 --> 00:53:55,810
So what else might that be?

1060
00:54:02,572 --> 00:54:06,436
AUDIENCE: Could it be the GST?

1061
00:54:06,436 --> 00:54:09,680
ELIZABETH NOLAN: Well, you
know that the GST/MS2 is there

1062
00:54:09,680 --> 00:54:14,030
because it was GSH that was
used to elute that whole thing.

1063
00:54:14,030 --> 00:54:18,890
So the ribosome and
the GST/MS2 fusion.

1064
00:54:18,890 --> 00:54:22,130
So I guess the question
is, just thinking what is

1065
00:54:22,130 --> 00:54:23,570
the size of the fusion protein?

1066
00:54:26,270 --> 00:54:28,670
And then how does that
compare to the ribosome?

1067
00:54:28,670 --> 00:54:30,710
And what does that mean
in terms of a shift

1068
00:54:30,710 --> 00:54:32,750
to a different percent sucrose?

1069
00:54:38,306 --> 00:54:41,380
So just what are possibilities?

1070
00:54:41,380 --> 00:54:45,220
We learned that the fusion
protein had some problems,

1071
00:54:45,220 --> 00:54:47,110
that it liked to aggregate.

1072
00:54:47,110 --> 00:54:49,180
Is it possible that contributes?

1073
00:54:49,180 --> 00:54:53,700
Is it possible that not all of
the 70S was dissociated, right?

1074
00:54:53,700 --> 00:54:56,680
So there is not a label for
where the 70S would come here.

1075
00:54:56,680 --> 00:54:59,980
These are just things to think
about when looking at the data

1076
00:54:59,980 --> 00:55:04,060
here and to look at
their explanation.

1077
00:55:04,060 --> 00:55:06,430
So is this good
news or bad news?

1078
00:55:06,430 --> 00:55:08,524
And is there a solution?

1079
00:55:08,524 --> 00:55:09,289
Yeah?

1080
00:55:09,289 --> 00:55:09,955
AUDIENCE: Sorry.

1081
00:55:09,955 --> 00:55:10,909
I had a question.

1082
00:55:10,909 --> 00:55:14,725
Since they're monitoring
the A280, so the protein

1083
00:55:14,725 --> 00:55:21,005
absorption, since the
50S would have the fusion

1084
00:55:21,005 --> 00:55:22,380
protein associated
with it still,

1085
00:55:22,380 --> 00:55:25,170
wouldn't that affect the A280?

1086
00:55:25,170 --> 00:55:27,120
Can we definitively
say that it's

1087
00:55:27,120 --> 00:55:29,550
actually depleted in
the 30S or is maybe

1088
00:55:29,550 --> 00:55:33,430
just the absorbance of the 50S
higher for the tagged protein

1089
00:55:33,430 --> 00:55:34,347
because of the fusion?

1090
00:55:34,347 --> 00:55:35,263
ELIZABETH NOLAN: Yeah.

1091
00:55:35,263 --> 00:55:36,660
So that's a good point, right?

1092
00:55:36,660 --> 00:55:40,230
The fusion protein is going to
have some absorbance defined

1093
00:55:40,230 --> 00:55:42,050
by its extinction coefficient.

1094
00:55:42,050 --> 00:55:45,030
And then the question is,
what is that in magnitude?

1095
00:55:45,030 --> 00:55:47,520
And how does that compare
to, say, the total extinction

1096
00:55:47,520 --> 00:55:49,860
coefficient of the 50S, right?

1097
00:55:49,860 --> 00:55:54,012
So there are a number of
ribosomal proteins, 20 to 30,

1098
00:55:54,012 --> 00:55:55,470
and we don't know
how many of those

1099
00:55:55,470 --> 00:56:00,110
got through this purification,
but they're there.

1100
00:56:00,110 --> 00:56:02,610
So that's how I would
go about analyzing that.

1101
00:56:02,610 --> 00:56:05,430
I actually don't know what
the relative absorbance

1102
00:56:05,430 --> 00:56:11,330
of the fusion protein to
the intact 50S ribosome is

1103
00:56:11,330 --> 00:56:15,088
but that's a caveat to
consider and could contribute.

1104
00:56:20,360 --> 00:56:21,547
So what do we think?

1105
00:56:21,547 --> 00:56:23,130
What are you going
to do for an assay?

1106
00:56:36,740 --> 00:56:39,490
If we were to set these
ribosomes up, the wild type

1107
00:56:39,490 --> 00:56:42,400
and the mutant one, and,
let's say, do a peptide bond

1108
00:56:42,400 --> 00:56:46,770
formation reaction, like what
they presented in the paper

1109
00:56:46,770 --> 00:56:48,680
as is, what would you expect?

1110
00:56:48,680 --> 00:56:51,160
Are you going to see the same
activity or less activity?

1111
00:56:55,160 --> 00:56:59,230
Working under the model that
we don't have enough 30S here

1112
00:56:59,230 --> 00:57:04,550
to fully form 70S ribosomes.

1113
00:57:04,550 --> 00:57:07,100
AUDIENCE: You'd
expect the 30S to act

1114
00:57:07,100 --> 00:57:09,970
like the limiting agent
in peptide bond formation

1115
00:57:09,970 --> 00:57:11,240
where they come together.

1116
00:57:11,240 --> 00:57:15,790
And you can only make as much
ribosome as you have 30S.

1117
00:57:15,790 --> 00:57:19,222
So you would get less
peptide bond formation.

1118
00:57:19,222 --> 00:57:20,180
ELIZABETH NOLAN: Right.

1119
00:57:20,180 --> 00:57:23,240
So you'd have fewer
70S assembled ribosomes

1120
00:57:23,240 --> 00:57:25,141
that can do translation.

1121
00:57:25,141 --> 00:57:25,640
Right.

1122
00:57:25,640 --> 00:57:27,501
So what was their
solution to this problem?

1123
00:57:31,349 --> 00:57:33,190
Or if you don't remember,
what would you do?

1124
00:57:50,800 --> 00:57:53,770
What happens if we don't have
enough of a given component?

1125
00:57:53,770 --> 00:57:57,890
We'll finish on this note.

1126
00:57:57,890 --> 00:58:00,160
AUDIENCE: You just
add more of the 30S.

1127
00:58:00,160 --> 00:58:02,290
ELIZABETH NOLAN: Yeah, right?

1128
00:58:02,290 --> 00:58:05,990
So you can imagine
adding in more 30s there.

1129
00:58:05,990 --> 00:58:09,100
We know how to purify
wild-type ribosomes,

1130
00:58:09,100 --> 00:58:11,140
can dissociate the
subunits and imagine

1131
00:58:11,140 --> 00:58:15,460
purifying 30S and adding that
into the reaction to have

1132
00:58:15,460 --> 00:58:17,920
the correct stochiometry there.

1133
00:58:17,920 --> 00:58:21,340
So that's exactly what
they did in their assays.

1134
00:58:21,340 --> 00:58:24,580
And so I encourage you to
take a look at the assays

1135
00:58:24,580 --> 00:58:27,040
they did for peptide
bond formation

1136
00:58:27,040 --> 00:58:29,500
and for peptide release.

1137
00:58:29,500 --> 00:58:33,610
And in the posted notes,
there's a schematic overview

1138
00:58:33,610 --> 00:58:36,310
of everything that was
done to get the components

1139
00:58:36,310 --> 00:58:37,330
of their syringes.

1140
00:58:37,330 --> 00:58:39,070
So these were quench
flow experiments,

1141
00:58:39,070 --> 00:58:41,470
like what you talked about
in recitation in prior weeks

1142
00:58:41,470 --> 00:58:43,570
and we talked about with EFP.

1143
00:58:43,570 --> 00:58:46,630
And there's also an example
where they used puromycin,

1144
00:58:46,630 --> 00:58:51,820
like what we saw with EFP,
in the experimental setup, so

1145
00:58:51,820 --> 00:58:55,710
that antibiotic that causes
change termination there.

1146
00:58:55,710 --> 00:58:58,990
And so what they found is that
these tagged ribosomes really

1147
00:58:58,990 --> 00:59:04,190
work quite well in terms of
the kinetic comparison there.

1148
00:59:04,190 --> 00:59:06,040
And they moved on
with this methodology

1149
00:59:06,040 --> 00:59:08,910
to use it to purify new
ribosomes for other studies.

1150
00:59:08,910 --> 00:59:12,370
So it turned out to be a
useful method for their lab,

1151
00:59:12,370 --> 00:59:15,070
and I would argue, what would be
a useful method for other labs,

1152
00:59:15,070 --> 00:59:19,570
based on the information they
presented in their paper there.

1153
00:59:19,570 --> 00:59:20,740
OK?

1154
00:59:20,740 --> 00:59:24,520
So you're off the hook, and
I'll see you in recitation

1155
00:59:24,520 --> 00:59:26,670
next week.