Transcript 3x8_Take_2

BioLogic
• We’re going to use
– The Quorum System
– Small RNAs
– 2-Hybrid systems (and a 3-Hybrid system)
Picture of sRNA system
Picture of the Lux Autoinducer
system
Hybrid Systems
1.
2.
3.
4.
A+B
C+D
E+F
X+Y+Z
B2H1A+B2H1B
B2H2A+B2H2B
B2H3A+B2H3B
B3H1A+B3H1B+B3H1C
Schematic of a 3-Hybrid System
Schematic of a 2-Hybrid System
TET
Gene, Autoinducer(s)
1
Gene 1
- - pLuxR
2
+ - -
Lux R
LuxI
pRhiR
3
- + -
- - +
tet R
X
RhlI
tet R
Y
?
+ + -
?
tet R
Z
Spot42
+ - +
- + +
+ + +
SgrS
D
F
SgrS
SgrS
CD
Spot42
Gene 6
SgrS
EF
Spot42
Gene 7
SgrS
XYZ
8
E
Gene 5
EF
7
A
SgrS
AB
CD
6
C
Gene 2
Gene 3
?
AB
5
B
pRhlI
RhlR
?
4
pLuxI
XYZ
Gene 8
Spot42
Gene 4
Small RNAs in E. coli
• We’re planning to use Spot 42 (which binds to the
RBS) and SgrS (which binds to the 5’ UTR and recruits
degradative enzymes) because there are professors
on campus using them successfully.
• We have access to strains of bacteria where
the endogenous Spot 42 and SgrS systems
have been knocked out.
• Protocols regarding working with sRNAs:
– Urban JH, Vogel J. Translational control and target recognition by Escherichia
coli small RNAs in vivo. Nucleic Acids Res. 2007;35(3):1018-37. Epub 2007 Jan 30.
PubMed PMID: 17264113; PubMed Central PMCID: PMC1807950.
Autoinducers
• We want to use autoinducers because of the
quick reaction time.
• We are planning to use LuxR and LuxI from
Vibrio fisheri. This uses the autoinducer N-(3Oxohexanoyl)-HSL.
• Also the RhlI and RhlR system from
Pseudomonas aeruginosa with the
autoinducer N-(butyryl)-HSL
• We have not characterized the final
autoinducer system yet.
Hybrid Systems
1.
2.
3.
4.
A+B
C+D
E+F
X+Y+Z
B2H1A+B2H1B
B2H2A+B2H2B
B2H3A+B2H3B
B3H1A+B3H1B+B3H1C
Schematic of a 3-Hybrid System
Schematic of a 2-Hybrid System
Hybrid Systems Promoters
B2H1A+B2H1B
10 bp
Zif269BS
63 bp
P(wk); weak Lac
Promoter from E coli
10 bp
B2H2A+B2H2B
TATA zifvar
B2H3A+B2H3B
P(wk); weak Lac
Promoter from E coli
10 bp
P53zifvar
B3H1A+B3H1B+B3H1C
63 bp
63 bp
P(wk); weak Lac
Promoter from E coli
10 bp
OL2
62 bp
P(wk); weak Lac
Promoter from E coli
B2H1
Ala-Ala-Ala Linker
1
248
257
278
B2H1A
E. Coli RNA Polymerase
Subunit A
(residues 1-248)
Yeast Gal4 protein
(residues 58-92)
AAAPVRTG Linker
1
89
113
B2H1B
Yeast Gal 11P
(residues 263-352)
*N341V mutation
Zif 268
(residues 327-421)
207
B2H2
Ala-Ala-Ala Linker
1
248
257
823
B2H2A
E. Coli RNA Polymerase
Subunit A
(residues 1-248)
GacS
(residues 253-819)
AAAPVRTG Linker
1
89
113
B2H2B
GacA
TFIIB (DBD)
B2H3
Ala-Ala-Ala Linker
1
248
257
B2H3A
E. Coli RNA Polymerase
Subunit A
(residues 1-248)
MavT
AAAPVRTG Linker
1
62
86
B2H3B
P53 (DBD)
MavU
(residues 1-62)
B3H1
B3H1A
FtsB
B3H1B
FtsL
B3H1C
FtsW
CI (DBD)
Pros
• It would be really cool and
have lots interesting, novel
aspects like the sRNA,
hybrid systems, and
autoinducer systems.
• A quick reaction time.
• We’d be introducing the
hybrid system as a logic
gate.
• With sRNAs and hybrid
system, you can create any
comination of gates.
Cons
• All the novel ideas makes it
hard and complex.
• Time consuming.
• We need to characterize a
third autoinducer.
• Each aspect of our project is
a project within itself.
• There is a lot of data that
we will need to reproduce.
Questions?!?!?
• Time:
– How much time would it take to make the 2:4?
– How much time would it then take to complete
the 3:8?
• How practical is it to assume that we’ll be able
to recreate the literature:
– sRNAs
– Hybrid Systems
– Autoinducers
• Are acyl-ACP and SAM naturally produced in E.
coli?
The End
Gene of Interest
PA1, PAL1, PA2
A 0 0 0
TET
tet
Luxpr
pLaxCl
B 1 0 0
Lux R
tet R
LAS R
tet R
Lux Q
tet R
AB
E 11 0
MicA
Gene B
OxyS
GcvB
Gene C
V
D
F
MicC
RhyB
Gene D
AB
AB
Spot 42
Gene F
MicA
SgrS
Gene G
W
RhyB
EF
MicC
VW
AB
GcvB
CD
EF
EF
H 1 1 1
SgrS
E
CD
MicA
G 0 1 1
Spot42
A
Gene E
CD
F 1 0 1
C
pLUX
pLux
D 0 0 1
B
pLAS
pLaS
C 0 1 0
Gene A
OxyS
VW
Gene H
MicA
Gene of Interest
PA1, PAL1, PA2
A 0 0 0
System using 2 small RNAs
TET
tet
Luxpr
pLaxCl
B 1 0 0
Lux R
tet R
LAS R
tet R
Lux Q
tet R
MicA
Spot 42
Gene C
V
D
F
Spot 42
Gene D
Spot 42
Gene F
Spot 42
EF
EF
H 1 1 1
E
CD
MicA
G 0 1 1
Gene B
A
Gene E
CD
F 1 0 1
Spot 42
AB
AB
E 11 0
C
pLUX
pLux
D 0 0 1
B
pLAS
pLaS
C 0 1 0
Gene A
MicA
Gene G
Spot 42
VW
AB
W
Gene H
Gene of Interest
PA1, PAL1, PA2
A 0 0 0
Same deal with a 3 Hybrid System
TET
tet
Luxpr
pLaxCl
B 1 0 0
Lux R
tet R
LAS R
B
Lux Q
Y
A
E
Spot 42
Gene C
tet R
Z
D
F
Spot 42
Gene D
AB
MicA
Gene E
Spot 42
CD
CD
F 1 0 1
MicA
Gene F
Spot 42
EF
EF
G 0 1 1
Spot 42 Gene B
tet R
AB
E 11 0
C
pLUX
pLux
D 0 0 1
X
pLAS
pLaS
C 0 1 0
Gene A
MicA
Gene G
Spot 42
XYZ
XYZ
H 1 1 1
Gene H
MicA
Small RNAs in E. coli
• All the ones in the following chart have a high
efficiency
• The following chart comes from “The Small
RNA Regulators of Escherichia Coli: Roles and
Mechanisms” by Susan Gottesman
• Protocalls regarding working with sRNAs: Urban JH,
Vogel J. Translational control and target recognition by Escherichia
coli small RNAs in vivo. Nucleic Acids Res. 2007;35(3):1018-37. Epub 2007 Jan 30.
PubMed PMID: 17264113; PubMed Central PMCID: PMC1807950.
• Another good Source: Regulatory RNAs in
Bacteria by Gisela Storz ;
http://www.sciencedirect.com.proxy2.library.uiuc.edu/science?_ob=ArticleURL&_udi=B6WSN-4VNHRSCB&_user=571676&_coverDate=02%2F20%2F2009&_rdoc=10&_fmt=high&_orig=browse&_srch=docinfo(%23toc%237051%232009%23998639995%23933091%23FLA%23display%23Volume)&_cdi=7051&_sort=d&_docancho
r=&_ct=22&_acct=C000029040&_version=1&_urlVersion=0&_userid=571676&md5=db7004c2567e64a29f9508281abc76ac
Category
Numbe
r
Examples
Size (nt) Mechanism/ Regulator
role
s/comme
nts
Reference
s
Hfqbinding,
antisense
22
DsrA
85
Stimulates
rpoS
Inhibits hns
Low
temp.,
LeuO
58, 75
RprA
105
Stimulates
rpoS
RcsC/B
phosphor
elay
59
OxyS
109
Anti-rpoS,
fhlA
OxyR
2,3
RyhB/SraI
90
Anti-sdh,
sodB
Fur
61
Spot 42
109
Anti-galK
CRP/cAMP 68
MicF
93
Anti-ompF
SoxR/S
22
MicC
108
Anti-ompC
Inverse to
MicF
19,19a
DicF
56
Anti-ftsZ
Phage
promoter
10
RyeA/SraC
275
Anti-RyeB
Unknown
100
Antisense
3
sRNAs that we’re going to use:
SgrS
http://www.ncbi.nlm.nih.gov.proxy2.library.uiuc.edu/pubmed/18042713?ordinalpos=1&itool=En
trezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVD
ocSum
Spot42
http://search.ebscohost.com/login.aspx?direct=true&db=aph&AN=14891380&site=ehost-live
(sign into the U of I library)
OxyS
http://www.sciencedirect.com.proxy2.library.uiuc.edu/science?_ob=MImg&_imagekey=B6WSN419B8BT-7C&_cdi=7051&_user=571676&_orig=browse&_coverDate=07%2F11%2F1997&_sk=999099998&
view=c&wchp=dGLzVzz-zSkzk&md5=d29bac7ead27fdc1b702edb28fcadd11&ie=/sdarticle.pdf
http://www.ncbi.nlm.nih.gov/pubmed/9230301?log$=activity
GcvB
http://web.ebscohost.com.proxy2.library.uiuc.edu/ehost/pdf?vid=2&hid=105&sid=a661093e1c90-4ffa-be77-2b4e7f4b00b5%40sessionmgr102#db=hch&AN=6012035
MicC
https://ms5.express.cites.uiuc.edu/wm/mail/fetch.html?urlid=75b51be9449ffa2eb80ed26874c1
e5b44&url=http%3A%2F%2Fwww.pubmedcentral.nih.gov.proxy2.library.uiuc.edu%2Farticlerend
er.fcgi%3Ftool%3Dpubmed%26pubmedid%3D15466019
RyhB
http://www.pnas.org/content/99/7/4620.full
MicA
Access the most recent version at doi:10.1101/gad.354405
Genes Dev. 2005 19: 2355-2366
Klas I. Udekwu, Fabien Darfeuille, Jörg Vogel, et al.
antisense RNA
Hfq-dependent regulation of OmpA synthesis is mediated by an
MicA secondarystructure and
binding
The part marked B in the upper left is the DNA
sequence for the MicC gene. The part marked A shows
the binding site of MicC.
RyhB
Figure 2 Complementarity between the
sdhCDAB operon and RyhB. Genes of the
sdhCDAB operon are shown in A. Lines
marked EM8 and EM9 show the position of the
oligonucleotide probes used for Northern blots
(Fig. 3). B shows the predicted interaction
between RyhB and the sdhCDAB sense
strand. The ribosome binding site for sdhD is
underlined. The start codon for sdhD is shown
underlined and in italics, and the stop codon
for sdhC is shown in gray.
OxyS
It negatively controls
oxidative stress
response within the
cell.
Spot 42
SgrS
GcvB
Quantification of Lux system
• http://www.pubmedcentral.nih.gov/picrender
.fcgi?artid=176701&blobtype=pdf (This is not
as applicable)
• http://www.sciencedirect.com.proxy2.library.
uiuc.edu/science?_ob=ArticleURL&_udi=B6W
BK4PRHJ6K2&_user=571676&_rdoc=1&_fmt=
&_orig=search&_sort=d&view=c&_acct=C000
029040&_version=1&_urlVersion=0&_userid=
571676&md5=ea6566137620b30f76b459cf25
2ad23a
– (Log into the U of I library online database first)
Efficiency of Autoinducers
They used 3-oxohexanoylhomoserine lactone
(OHHL)
Kinetics of Autoinducers
We’re using a non-feedback system, so look at the triangles and the green.
Autoinducers
• 3OC6HSL is AI-1 which interacts with LuxR to
activate pLuxCl, which activates Luxpr.
• 3OC12HSL is PAI-1, which interacts with LasR
to activate pLAS, which activates pLAS
• Furanosyl borate diester is AI-2, which
interacts with LuxQ to activate pLux, which
activates pLuxpr.
•LasR
PAI 1
3OC12HSL
LuxR
AI-1
3OC6HSL
LuxQ
AI-2
Furanosyl borate
diester
Hybrid Systems
1.
2.
3.
4.
5.
AB
CD
EF
VW
XYZ
B2H1A+B2H1B
B2H2A+B2H2B
B2H3A+B2H3B
B2H4A+B2H4B
B3H1A+B3H1B+B3H1C
Schematic of a 2-Hybrid System
2
Zif
1
α
RNA Pol
Promoter
Yeast 2-Hybrid System
1
2
α
Zif
RNA Pol
P(wk); weak Lac
Promoter from E coli
10 bp
10 bp
10 bp
63 bp
P(wk); weak Lac
Promoter from E coli
B2H1A; αGal4 protein
Ala-Ala-Ala Linker
1
248
E. Coli RNA Polymerase
Subunit A
(residues 1-248)
257
278
Yeast Gal4 protein
(residues 58-92)
On pACYC184 – derived pACL- αGal4 protein  1 PTG-inducible 1pp/lacUr5
B2H1B; Gal 11P – Zif 123
AAAPVRTG Linker
1
89
Yeast Gal 11P
(residues 263-352)
*N341V mutation
113
Zif 268
(residues 327-421)
On pBR-GP-2123  Phagemid
207