1
00:00:25,589 --> 00:00:23,429
so chaotropic environments are hyper

2
00:00:25,830 --> 00:00:25,599
saline brines with a high concentration

3
00:00:29,189 --> 00:00:25,840
of

4
00:00:31,029 --> 00:00:29,199
ketotropes broadly speaking ko trips can

5
00:00:32,709 --> 00:00:31,039
be thought of as order breakers which

6
00:00:35,350 --> 00:00:32,719
disrupt h-bonding

7
00:00:37,430 --> 00:00:35,360
um in bulk solution these will denature

8
00:00:40,069 --> 00:00:37,440
biological macromolecules

9
00:00:41,030 --> 00:00:40,079
uh leading to cell death and high enough

10
00:00:43,270 --> 00:00:41,040
concentration

11
00:00:44,069 --> 00:00:43,280
so a common keotrope environmental

12
00:00:46,069 --> 00:00:44,079
chaotrope would be

13
00:00:47,990 --> 00:00:46,079
chloride so in a very high concentration

14
00:00:50,229 --> 00:00:48,000
of chloride this will inhibit

15
00:00:51,590 --> 00:00:50,239
cellular function via protein

16
00:00:54,630 --> 00:00:51,600
denaturation

17
00:00:56,389 --> 00:00:54,640
and enzyme inhibition and to a certain

18
00:00:57,990 --> 00:00:56,399
concentration and for example in this

19
00:01:00,470 --> 00:00:58,000
magnesium chloride brine

20
00:01:01,110 --> 00:01:00,480
southern california being largely

21
00:01:04,469 --> 00:01:01,120
considered

22
00:01:06,870 --> 00:01:04,479
sterile in these upper limits however

23
00:01:07,670 --> 00:01:06,880
there's some speculative evidence of

24
00:01:10,830 --> 00:01:07,680
active life

25
00:01:12,870 --> 00:01:10,840
within magnesium chloride saturated

26
00:01:15,590 --> 00:01:12,880
dhabs

27
00:01:17,190 --> 00:01:15,600
deep sea hyper saline and oxide basins

28
00:01:17,749 --> 00:01:17,200
these are noxic brine environments that

29
00:01:19,590 --> 00:01:17,759
are found

30
00:01:21,510 --> 00:01:19,600
in benthic depressions generally

31
00:01:22,870 --> 00:01:21,520
enclosed seas such as the eastern

32
00:01:26,789 --> 00:01:22,880
mediterranean

33
00:01:28,149 --> 00:01:26,799
and the red sea so there's a few dozen d

34
00:01:30,230 --> 00:01:28,159
habs that have been discovered

35
00:01:31,350 --> 00:01:30,240
most of them are sodium chloride

36
00:01:33,350 --> 00:01:31,360
saturated with

37
00:01:34,950 --> 00:01:33,360
um a swath of evidence showing active

38
00:01:36,789 --> 00:01:34,960
microbial communities

39
00:01:39,429 --> 00:01:36,799
while only three have been shown to be

40
00:01:42,310 --> 00:01:39,439
magnesium chloride dominated

41
00:01:43,990 --> 00:01:42,320
so within this interface layer so in

42
00:01:46,149 --> 00:01:44,000
this figure this is just showing a

43
00:01:47,429 --> 00:01:46,159
basic d hem so an ancient evaporate

44
00:01:49,030 --> 00:01:47,439
which then dissolves

45
00:01:50,469 --> 00:01:49,040
forming a brine that collects an

46
00:01:52,149 --> 00:01:50,479
adventic depression

47
00:01:53,749 --> 00:01:52,159
here we have a two to three meter

48
00:01:55,749 --> 00:01:53,759
interface layer where you have an

49
00:01:56,230 --> 00:01:55,759
extremely steep chemokline going from

50
00:01:59,190 --> 00:01:56,240
about

51
00:01:59,749 --> 00:01:59,200
zero millimolar um sorry zero molar all

52
00:02:02,230 --> 00:01:59,759
the way

53
00:02:03,749 --> 00:02:02,240
upwards of five molar magnesium chloride

54
00:02:05,030 --> 00:02:03,759
within the brine and member

55
00:02:07,270 --> 00:02:05,040
and that only two to three meter

56
00:02:09,469 --> 00:02:07,280
interface so so the current magnesium

57
00:02:12,710 --> 00:02:09,479
chloride limit of life is around

58
00:02:14,470 --> 00:02:12,720
2.3 some studies going as far to say

59
00:02:15,270 --> 00:02:14,480
that it's around three more in some of

60
00:02:18,869 --> 00:02:15,280
these

61
00:02:19,830 --> 00:02:18,879
dehabs whereas again as i mentioned this

62
00:02:21,990 --> 00:02:19,840
brine being

63
00:02:23,030 --> 00:02:22,000
four to five molar magnesium chloride

64
00:02:25,670 --> 00:02:23,040
and there's some

65
00:02:28,150 --> 00:02:25,680
speculative evidence in the cryosprine

66
00:02:29,670 --> 00:02:28,160
that shows sulfate reduction

67
00:02:32,790 --> 00:02:29,680
so this was just to really highlight

68
00:02:34,790 --> 00:02:32,800
this limit being around to it 2.3

69
00:02:36,949 --> 00:02:34,800
and then the brine itself being the 4

70
00:02:38,790 --> 00:02:36,959
fortified molar which is significantly

71
00:02:40,790 --> 00:02:38,800
more stressful

72
00:02:42,869 --> 00:02:40,800
the reason why we're interested in

73
00:02:43,910 --> 00:02:42,879
dehabs especially magnesium core id

74
00:02:46,309 --> 00:02:43,920
heads

75
00:02:47,910 --> 00:02:46,319
uh that they share some similarities in

76
00:02:50,869 --> 00:02:47,920
the hypothesized

77
00:02:53,030 --> 00:02:50,879
brine lenses on europa as well as some

78
00:02:55,509 --> 00:02:53,040
perchlorate brines on mars

79
00:02:57,030 --> 00:02:55,519
and then subsurface oceans of enceladus

80
00:02:58,710 --> 00:02:57,040
so we get this nice window to

81
00:03:01,509 --> 00:02:58,720
extraterrestrial brines

82
00:03:03,030 --> 00:03:01,519
via the scope of the dhabs and the

83
00:03:04,470 --> 00:03:03,040
motivation of this project that i'll be

84
00:03:06,949 --> 00:03:04,480
talking about today

85
00:03:09,030 --> 00:03:06,959
is to unders better understand micro

86
00:03:11,190 --> 00:03:09,040
microbial adaptation to magnesium

87
00:03:12,869 --> 00:03:11,200
chloride brines as the genes associated

88
00:03:14,869 --> 00:03:12,879
with life in these environments

89
00:03:15,990 --> 00:03:14,879
are virtually unknown there's some

90
00:03:17,910 --> 00:03:16,000
strategies that are

91
00:03:19,110 --> 00:03:17,920
associated with sodium chloride

92
00:03:21,190 --> 00:03:19,120
adaptation

93
00:03:22,309 --> 00:03:21,200
but specific to magnesium chloride there

94
00:03:24,070 --> 00:03:22,319
are some holes

95
00:03:25,350 --> 00:03:24,080
and the limits of life and magnesium

96
00:03:27,509 --> 00:03:25,360
chloride brines

97
00:03:28,550 --> 00:03:27,519
are poorly defined there's there's a lot

98
00:03:31,110 --> 00:03:28,560
of speculation

99
00:03:32,869 --> 00:03:31,120
of whether they're in a higher

100
00:03:34,149 --> 00:03:32,879
concentration brine that four to five

101
00:03:35,509 --> 00:03:34,159
molar zone

102
00:03:37,830 --> 00:03:35,519
so the study i'll talk about today is

103
00:03:40,070 --> 00:03:37,840
evolution of e coli to increase

104
00:03:41,830 --> 00:03:40,080
its magnesium chloride stress so the

105
00:03:44,309 --> 00:03:41,840
goal of this project was to engineer

106
00:03:46,390 --> 00:03:44,319
kyoto e coli strains using adaptive

107
00:03:48,949 --> 00:03:46,400
laboratory evolution hereby known as

108
00:03:50,550 --> 00:03:48,959
all for short with the purpose to

109
00:03:52,550 --> 00:03:50,560
identify genes that confer

110
00:03:54,630 --> 00:03:52,560
magnesium chloride tolerance with the

111
00:03:55,589 --> 00:03:54,640
big why to have this improved molecular

112
00:03:57,350 --> 00:03:55,599
understanding

113
00:03:58,710 --> 00:03:57,360
of the limits of life in chaotropic

114
00:04:01,509 --> 00:03:58,720
brines

115
00:04:03,110 --> 00:04:01,519
so adaptive laboratory evolution or ale

116
00:04:05,190 --> 00:04:03,120
again is based upon principles of

117
00:04:06,710 --> 00:04:05,200
darwinian evolution to develop evolved

118
00:04:07,910 --> 00:04:06,720
microbial strains

119
00:04:09,589 --> 00:04:07,920
so you can think of you'd have your

120
00:04:10,309 --> 00:04:09,599
original population of cells in your

121
00:04:12,630 --> 00:04:10,319
test tube

122
00:04:14,070 --> 00:04:12,640
if you increase a given stressor in my

123
00:04:15,750 --> 00:04:14,080
case magnesium chloride

124
00:04:17,670 --> 00:04:15,760
you'll have some rare survivors that

125
00:04:19,830 --> 00:04:17,680
might have a better adaptation

126
00:04:21,030 --> 00:04:19,840
just through random mutation to the

127
00:04:24,070 --> 00:04:21,040
changing environment

128
00:04:25,909 --> 00:04:24,080
this will then result in a population of

129
00:04:26,790 --> 00:04:25,919
cells that are slightly more tolerant to

130
00:04:28,310 --> 00:04:26,800
the stressor

131
00:04:30,550 --> 00:04:28,320
and as you keep going down the line

132
00:04:32,550 --> 00:04:30,560
you'll have more and more rare survivors

133
00:04:35,350 --> 00:04:32,560
until you eventually have a stress

134
00:04:37,590 --> 00:04:35,360
resistant lineage that is nearly ident

135
00:04:39,909 --> 00:04:37,600
genetically identical to that of the

136
00:04:43,189 --> 00:04:39,919
starting strain

137
00:04:44,710 --> 00:04:43,199
so once i completed my evolution

138
00:04:45,909 --> 00:04:44,720
experiment over a number of months i

139
00:04:47,510 --> 00:04:45,919
could then

140
00:04:50,150 --> 00:04:47,520
elucidate these mutations using a

141
00:04:52,150 --> 00:04:50,160
bioinformatics pipeline known as briseq

142
00:04:54,390 --> 00:04:52,160
so this involves taking the ancestral

143
00:04:56,550 --> 00:04:54,400
strain as well as your evolve strain

144
00:04:57,990 --> 00:04:56,560
throwing that through a uh sequencing

145
00:05:00,310 --> 00:04:58,000
company so i i went through

146
00:05:01,590 --> 00:05:00,320
illumina platform and then plugging into

147
00:05:03,590 --> 00:05:01,600
brie seek which will then

148
00:05:05,510 --> 00:05:03,600
output a user summary file that

149
00:05:07,510 --> 00:05:05,520
essentially compares

150
00:05:08,550 --> 00:05:07,520
the ancestral genome to that of the

151
00:05:10,390 --> 00:05:08,560
evolved genome

152
00:05:11,990 --> 00:05:10,400
and identifies where mutations have

153
00:05:13,350 --> 00:05:12,000
occurred throughout the course of your

154
00:05:15,590 --> 00:05:13,360
ale experiment

155
00:05:17,909 --> 00:05:15,600
so the brief method here was to grow e

156
00:05:18,790 --> 00:05:17,919
coli and lb and three lineages shown

157
00:05:21,189 --> 00:05:18,800
here

158
00:05:23,830 --> 00:05:21,199
incubating them checking for cfus as

159
00:05:25,749 --> 00:05:23,840
well as phenotype on auger plates

160
00:05:27,350 --> 00:05:25,759
check tracking their ods over time and

161
00:05:29,029 --> 00:05:27,360
storing some in -80

162
00:05:31,110 --> 00:05:29,039
and again at every single subsequent

163
00:05:32,310 --> 00:05:31,120
transfer i'm increasing magnesium

164
00:05:34,550 --> 00:05:32,320
chloride concentration

165
00:05:36,150 --> 00:05:34,560
and repeating the process so after about

166
00:05:39,670 --> 00:05:36,160
200 generations

167
00:05:42,390 --> 00:05:39,680
i saw nearly a um

168
00:05:43,990 --> 00:05:42,400
tolerance growth sorry growth tolerance

169
00:05:47,110 --> 00:05:44,000
that nearly doubled

170
00:05:48,870 --> 00:05:47,120
through the course of ale from a macro

171
00:05:52,629 --> 00:05:48,880
perspective just looking at the cells

172
00:05:54,310 --> 00:05:52,639
phenotypically the ancestral strain

173
00:05:56,390 --> 00:05:54,320
form you know just a classic dense

174
00:05:58,390 --> 00:05:56,400
here's a classic dense e coli culture

175
00:06:00,870 --> 00:05:58,400
and lb whereas the evolve strains were

176
00:06:01,670 --> 00:06:00,880
forming this biofilm this large macro

177
00:06:03,830 --> 00:06:01,680
structure

178
00:06:05,749 --> 00:06:03,840
that was actually tying itself in knots

179
00:06:07,830 --> 00:06:05,759
from this image here so all the cells

180
00:06:08,870 --> 00:06:07,840
really aggregating and clumping up close

181
00:06:11,830 --> 00:06:08,880
together

182
00:06:13,749 --> 00:06:11,840
i also noticed a corresponding decrease

183
00:06:17,510 --> 00:06:13,759
in cell density and a

184
00:06:20,950 --> 00:06:19,350
so after the course of ale to confirm

185
00:06:22,629 --> 00:06:20,960
that i'm actually getting the phenotype

186
00:06:24,390 --> 00:06:22,639
of increased growth magnesium chloride

187
00:06:26,790 --> 00:06:24,400
so on the left here we have

188
00:06:28,870 --> 00:06:26,800
um growth of e coli just in zero

189
00:06:31,990 --> 00:06:28,880
millimolar magnesium chloride lb

190
00:06:34,469 --> 00:06:32,000
lb is just a rich protein broth that's

191
00:06:35,510 --> 00:06:34,479
commonly used for mesophiles such as e

192
00:06:38,469 --> 00:06:35,520
coli

193
00:06:39,510 --> 00:06:38,479
so in red i have the ancestor in green

194
00:06:41,189 --> 00:06:39,520
and blue i have the b

195
00:06:43,909 --> 00:06:41,199
and the c lineage these are the two

196
00:06:47,909 --> 00:06:43,919
evolved lineage after the chorus of ale

197
00:06:49,990 --> 00:06:47,919
i have od 600 on the the y-axis which is

198
00:06:50,629 --> 00:06:50,000
just a measure of optical density so

199
00:06:54,469 --> 00:06:50,639
this is

200
00:06:57,670 --> 00:06:54,479
in a culture

201
00:06:58,710 --> 00:06:57,680
with time on the x in hours and what i

202
00:07:01,430 --> 00:06:58,720
found was that

203
00:07:01,990 --> 00:07:01,440
the evolved strains had a slightly

204
00:07:03,510 --> 00:07:02,000
depressed

205
00:07:06,230 --> 00:07:03,520
growth rate compared to that of the

206
00:07:08,150 --> 00:07:06,240
ancestor whereas

207
00:07:09,589 --> 00:07:08,160
growing them at an elevated magnesium

208
00:07:11,749 --> 00:07:09,599
chloride concentration

209
00:07:13,510 --> 00:07:11,759
the ancestor never achieved growth as

210
00:07:13,990 --> 00:07:13,520
you can see here here by this flat red

211
00:07:17,990 --> 00:07:14,000
line

212
00:07:20,790 --> 00:07:18,000
reaching a significant

213
00:07:21,589 --> 00:07:20,800
cell density um with nearly similar

214
00:07:25,749 --> 00:07:21,599
growth rates

215
00:07:27,350 --> 00:07:25,759
at 375 millimolar magnesium chloride

216
00:07:28,790 --> 00:07:27,360
i was curious to see how the strains

217
00:07:29,270 --> 00:07:28,800
respond to other salts to see if there

218
00:07:31,589 --> 00:07:29,280
was a more

219
00:07:33,990 --> 00:07:31,599
universal chaotropic sorry more

220
00:07:34,469 --> 00:07:34,000
universal solute stress tolerance or if

221
00:07:36,469 --> 00:07:34,479
their

222
00:07:37,909 --> 00:07:36,479
adaptation was very specific to that of

223
00:07:39,589 --> 00:07:37,919
magnesium chloride

224
00:07:42,150 --> 00:07:39,599
i noticed that obviously the evolved

225
00:07:44,469 --> 00:07:42,160
strain is more ko tolerant

226
00:07:46,309 --> 00:07:44,479
in sodium chloride we saw that the

227
00:07:47,830 --> 00:07:46,319
greatest taunt was actually surprisingly

228
00:07:49,510 --> 00:07:47,840
with the ancestral strain which is

229
00:07:50,550 --> 00:07:49,520
probably indicating a very specific

230
00:07:52,710 --> 00:07:50,560
adaptation

231
00:07:54,469 --> 00:07:52,720
within magnesium sulfate brines again

232
00:07:58,550 --> 00:07:54,479
another environmentally

233
00:08:01,749 --> 00:07:58,560
relevant brine we're seeing the same

234
00:08:04,150 --> 00:08:01,759
trend of just a slightly depressed uh

235
00:08:05,670 --> 00:08:04,160
cell density so the blues being the

236
00:08:07,189 --> 00:08:05,680
evolved lineages

237
00:08:08,950 --> 00:08:07,199
whereas the ancestor growing to much

238
00:08:09,909 --> 00:08:08,960
higher cell densities and magnesium

239
00:08:12,309 --> 00:08:09,919
sulfate

240
00:08:13,270 --> 00:08:12,319
what was great to see is with um calcium

241
00:08:15,749 --> 00:08:13,280
chloride

242
00:08:18,150 --> 00:08:15,759
um we saw the same trend with that of

243
00:08:21,189 --> 00:08:18,160
magnesium chloride so we're seeing

244
00:08:22,150 --> 00:08:21,199
strong growth in calcium chloride at 350

245
00:08:24,629 --> 00:08:22,160
millimolar

246
00:08:26,390 --> 00:08:24,639
with that of the evolved lineage evolved

247
00:08:28,150 --> 00:08:26,400
lineage so the blue

248
00:08:30,150 --> 00:08:28,160
or as the ancestor never achieving

249
00:08:31,350 --> 00:08:30,160
growth so this told us our

250
00:08:33,909 --> 00:08:31,360
interpretation was that

251
00:08:36,790 --> 00:08:33,919
the cells have a very specific chloride

252
00:08:40,230 --> 00:08:39,829
um upon analysis of the breseq data so

253
00:08:43,509 --> 00:08:40,240
again

254
00:08:45,430 --> 00:08:43,519
comparing the two genomes the ancestral

255
00:08:47,190 --> 00:08:45,440
as well as the evolved lineages

256
00:08:49,430 --> 00:08:47,200
the two mutations that were shared

257
00:08:51,829 --> 00:08:49,440
amongst the evolved strains b

258
00:08:53,190 --> 00:08:51,839
and c were involved in the men's sea

259
00:08:54,470 --> 00:08:53,200
gene which is involved in the production

260
00:08:56,389 --> 00:08:54,480
of metaquinoids

261
00:08:57,990 --> 00:08:56,399
these have a role in electron transport

262
00:09:00,630 --> 00:08:58,000
in the cell membrane

263
00:09:01,030 --> 00:09:00,640
and ubiquinone has been previously shown

264
00:09:05,350 --> 00:09:01,040
to

265
00:09:07,430 --> 00:09:05,360
certain bacteria

266
00:09:09,190 --> 00:09:07,440
so the mnc gene we need to further

267
00:09:10,790 --> 00:09:09,200
elucidate this and really

268
00:09:12,470 --> 00:09:10,800
dive deep into literature to have a

269
00:09:13,269 --> 00:09:12,480
better understanding but just from first

270
00:09:15,509 --> 00:09:13,279
glance

271
00:09:18,630 --> 00:09:15,519
there's some previous research that

272
00:09:20,829 --> 00:09:18,640
suggests that this mensee mutation

273
00:09:22,230 --> 00:09:20,839
would confer and increase osmotic

274
00:09:24,630 --> 00:09:22,240
tolerance

275
00:09:27,110 --> 00:09:24,640
and the other mutation that was shared

276
00:09:29,269 --> 00:09:27,120
was in the rcs system so the rcs genes

277
00:09:29,910 --> 00:09:29,279
regulate clonic acid production also

278
00:09:32,790 --> 00:09:29,920
known as

279
00:09:34,630 --> 00:09:32,800
eps eps is this negatively charged

280
00:09:36,150 --> 00:09:34,640
polysaccharide that forms a protective

281
00:09:38,550 --> 00:09:36,160
sheath around the cell

282
00:09:40,550 --> 00:09:38,560
previously shown to desiccate sorry to

283
00:09:43,269 --> 00:09:40,560
protect cells from desiccation

284
00:09:44,150 --> 00:09:43,279
as well as osmotic stress so this brief

285
00:09:47,829 --> 00:09:44,160
figure

286
00:09:49,750 --> 00:09:47,839
um from this paper indicated here

287
00:09:51,750 --> 00:09:49,760
we have the environmental stimuli so if

288
00:09:53,990 --> 00:09:51,760
this is your cell membrane this is

289
00:09:55,990 --> 00:09:54,000
inside the cell this is the the

290
00:09:57,750 --> 00:09:56,000
peptidoglycan layer

291
00:10:00,150 --> 00:09:57,760
we would have an environmental stimuli

292
00:10:02,710 --> 00:10:00,160
represented by the black lightning bolts

293
00:10:03,750 --> 00:10:02,720
the rcs system is then activated

294
00:10:06,790 --> 00:10:03,760
phosphorus

295
00:10:08,710 --> 00:10:06,800
this phosphorylation cascade resulted in

296
00:10:10,870 --> 00:10:08,720
the production of eps which is this

297
00:10:13,030 --> 00:10:10,880
capsule indicated so then the eps would

298
00:10:13,990 --> 00:10:13,040
then be exuded outside of the cell and

299
00:10:17,190 --> 00:10:14,000
then protecting

300
00:10:18,150 --> 00:10:17,200
it from the osmotic stress so we

301
00:10:22,790 --> 00:10:18,160
identified

302
00:10:25,110 --> 00:10:22,800
rcsd and rcsb gene mutations

303
00:10:25,910 --> 00:10:25,120
the hypothesis right now simply put is

304
00:10:28,069 --> 00:10:25,920
just that the

305
00:10:29,990 --> 00:10:28,079
um evolve strain is probably producing

306
00:10:31,509 --> 00:10:30,000
excess eps so the magnesium chloride is

307
00:10:34,550 --> 00:10:31,519
perturbing the cell membrane

308
00:10:37,030 --> 00:10:34,560
activating the rcs fossil relay so the

309
00:10:38,630 --> 00:10:37,040
mutated rcs genes might be upregulating

310
00:10:40,470 --> 00:10:38,640
eps production

311
00:10:42,630 --> 00:10:40,480
and there's previous evidence showing

312
00:10:44,310 --> 00:10:42,640
the properties of eps binding heavy

313
00:10:47,750 --> 00:10:44,320
metals such as calcium

314
00:10:49,590 --> 00:10:47,760
or sorry not calcium zinc or arsenic

315
00:10:52,069 --> 00:10:49,600
so it's possible that this eps also has

316
00:10:53,590 --> 00:10:52,079
a magnesium binding effect

317
00:10:55,269 --> 00:10:53,600
and again this being consistent with

318
00:10:56,710 --> 00:10:55,279
that snotty phenotype of just this

319
00:11:01,430 --> 00:10:56,720
really large molecular

320
00:11:03,910 --> 00:11:01,440
sorry macro structure in the test tubes

321
00:11:05,430 --> 00:11:03,920
so now that we've seen the in silico

322
00:11:07,269 --> 00:11:05,440
data so the breeze data

323
00:11:08,790 --> 00:11:07,279
how do we find that smoking gun how do

324
00:11:10,710 --> 00:11:08,800
we determine if these genes

325
00:11:12,710 --> 00:11:10,720
actually confer magnesium chloride

326
00:11:15,670 --> 00:11:12,720
tolerance and this is now being done

327
00:11:17,750 --> 00:11:15,680
via genetics i'm using the lambda red

328
00:11:19,670 --> 00:11:17,760
system so i've successfully knocked out

329
00:11:21,350 --> 00:11:19,680
the genes of interest

330
00:11:24,389 --> 00:11:21,360
for the purpose of this talk i'll just

331
00:11:25,829 --> 00:11:24,399
talk about the rcsd gene

332
00:11:27,670 --> 00:11:25,839
and the lambda red system involves

333
00:11:29,670 --> 00:11:27,680
creating pcr products that have an

334
00:11:31,829 --> 00:11:29,680
antibiotic resistant cassette

335
00:11:34,069 --> 00:11:31,839
that will share homology to a target

336
00:11:37,430 --> 00:11:34,079
region so if the yellow

337
00:11:38,790 --> 00:11:37,440
indicates the homologous region of the

338
00:11:40,230 --> 00:11:38,800
target cell so like this would be the

339
00:11:41,190 --> 00:11:40,240
whole genome of the target cell for

340
00:11:43,430 --> 00:11:41,200
example

341
00:11:45,430 --> 00:11:43,440
i'm creating this linear this double

342
00:11:48,069 --> 00:11:45,440
stranded pcr product so i have my

343
00:11:51,030 --> 00:11:48,079
antibiotic resistant cassette here

344
00:11:52,790 --> 00:11:51,040
with the region of homology through

345
00:11:56,150 --> 00:11:52,800
recombination

346
00:11:59,030 --> 00:11:56,160
uh the antibiotic resistant cassette

347
00:12:00,790 --> 00:11:59,040
will replace itself with that of the the

348
00:12:01,509 --> 00:12:00,800
gene of interest so you'll excise the

349
00:12:03,350 --> 00:12:01,519
rcsd

350
00:12:06,150 --> 00:12:03,360
gene and these knockouts that then

351
00:12:09,269 --> 00:12:06,160
appear as drug-resistant colonies

352
00:12:11,590 --> 00:12:09,279
i then verified through pcr that i'm

353
00:12:12,790 --> 00:12:11,600
amplifying the rcsd gene product with my

354
00:12:15,990 --> 00:12:12,800
starting stream

355
00:12:17,430 --> 00:12:16,000
and i'm not amplifying the rcsd gene

356
00:12:19,670 --> 00:12:17,440
product with my knockouts

357
00:12:21,030 --> 00:12:19,680
so indicated here with the no

358
00:12:22,470 --> 00:12:21,040
amplification

359
00:12:24,069 --> 00:12:22,480
furthermore i just want to double check

360
00:12:25,990 --> 00:12:24,079
that i'm actually in the right spot so i

361
00:12:28,310 --> 00:12:26,000
designed primers that would

362
00:12:30,150 --> 00:12:28,320
design primers that would show this

363
00:12:33,030 --> 00:12:30,160
final construct

364
00:12:34,710 --> 00:12:33,040
to indicate that the antibiotic

365
00:12:35,910 --> 00:12:34,720
resistant cassette has been inserted in

366
00:12:37,509 --> 00:12:35,920
the correct spot

367
00:12:39,430 --> 00:12:37,519
in the starting strain not seeing that

368
00:12:41,990 --> 00:12:39,440
amplification whereas the knockout

369
00:12:43,670 --> 00:12:42,000
and also through sequencing showing that

370
00:12:44,949 --> 00:12:43,680
we actually have this correct final

371
00:12:46,310 --> 00:12:44,959
construct

372
00:12:49,030 --> 00:12:46,320
so the future work is to create this

373
00:12:50,949 --> 00:12:49,040
library as i've almost completed of all

374
00:12:51,750 --> 00:12:50,959
the mutations that i've identified via

375
00:12:55,190 --> 00:12:51,760
greasy

376
00:12:57,590 --> 00:12:55,200
to create double knockouts so to

377
00:12:58,629 --> 00:12:57,600
remove maybe the men's sea gene the rcsb

378
00:13:01,430 --> 00:12:58,639
gene meant

379
00:13:03,509 --> 00:13:01,440
rcsd gene removing those in tandem to

380
00:13:04,310 --> 00:13:03,519
identify the changes magnesium chloride

381
00:13:06,470 --> 00:13:04,320
tolerance

382
00:13:07,350 --> 00:13:06,480
and lastly would be the the best

383
00:13:09,829 --> 00:13:07,360
scenario

384
00:13:11,030 --> 00:13:09,839
would be to recreate the exact mutations

385
00:13:13,350 --> 00:13:11,040
that were identified

386
00:13:15,350 --> 00:13:13,360
via brie c and from there once i have

387
00:13:17,590 --> 00:13:15,360
this library constructed i can determine

388
00:13:20,949 --> 00:13:17,600
the ko tolerance of my mutants

389
00:13:21,990 --> 00:13:20,959
um which would then tell me exactly what

390
00:13:23,750 --> 00:13:22,000
the story is of

391
00:13:25,670 --> 00:13:23,760
how these genes are affecting the

392
00:13:26,629 --> 00:13:25,680
magnesium chloride tolerance of this e

393
00:13:29,269 --> 00:13:26,639
coli

394
00:13:30,870 --> 00:13:29,279
and for the purpose of this talk um

395
00:13:32,550 --> 00:13:30,880
obviously we're all interested

396
00:13:34,150 --> 00:13:32,560
in the halophiles are really interested

397
00:13:35,829 --> 00:13:34,160
in these

398
00:13:37,990 --> 00:13:35,839
these critters that have much more

399
00:13:40,550 --> 00:13:38,000
significance to an astrobiological

400
00:13:42,710 --> 00:13:40,560
astrobiological context and i want to

401
00:13:44,069 --> 00:13:42,720
mention that i'm continuing these same

402
00:13:46,069 --> 00:13:44,079
ale experiments with that of

403
00:13:47,030 --> 00:13:46,079
halobacterium salinarum a model

404
00:13:48,870 --> 00:13:47,040
halophile

405
00:13:51,750 --> 00:13:48,880
as well as methanohalophilus portico

406
00:13:54,069 --> 00:13:51,760
lenses so the same workflow

407
00:13:56,150 --> 00:13:54,079
of ale to increasing magnesium chloride

408
00:13:57,110 --> 00:13:56,160
tolerance with those model halophiles

409
00:13:58,949 --> 00:13:57,120
that i'm

410
00:14:01,350 --> 00:13:58,959
very much looking forward to i'm sharing

411
00:14:02,550 --> 00:14:01,360
the results with soon

412
00:14:04,470 --> 00:14:02,560
and lastly just wanted to make some

413
00:14:06,790 --> 00:14:04,480
acknowledgments to the bartlett lab

414
00:14:08,389 --> 00:14:06,800
um this is my lab at uc san diego at

415
00:14:09,269 --> 00:14:08,399
scripps institution of oceanography

416
00:14:11,590 --> 00:14:09,279
here's the

417
00:14:13,269 --> 00:14:11,600
lab pictured here um as well as oceans

418
00:14:15,189 --> 00:14:13,279
across space and time this is a

419
00:14:17,430 --> 00:14:15,199
recently awarded nasa project that i'm

420
00:14:18,949 --> 00:14:17,440
funded under um eric allen one of my

421
00:14:20,470 --> 00:14:18,959
committee members who's been extremely

422
00:14:23,750 --> 00:14:20,480
helpful in developing uh

423
00:14:25,189 --> 00:14:23,760
the knockout procedure uh a big shout

424
00:14:27,110 --> 00:14:25,199
out to marco allen for sharing

425
00:14:28,710 --> 00:14:27,120
his relevant strains with me and then

426
00:14:32,150 --> 00:14:28,720
benjamin klempe

427
00:14:32,949 --> 00:14:32,160
a great friend and graduate student that

428
00:14:35,110 --> 00:14:32,959
we've been

429
00:14:36,550 --> 00:14:35,120
i've been working very closely with and

430
00:14:38,949 --> 00:14:36,560
lastly jeff bowman