BioLogic

1
BioLogic

• Were going to use
• The Quorum System
• Small RNAs
• 2-Hybrid systems (and a 3-Hybrid system)

2
Picture of sRNA system
3
Picture of the Lux Autoinducer system
4
Hybrid Systems

1. AB B2H1AB2H1B
2. CD B2H2AB2H2B
3. EF B2H3AB2H3B
4. XYZ B3H1AB3H1BB3H1C

Schematic of a 3-Hybrid System
Schematic of a 2-Hybrid System
5
TET
Gene, Autoinducer(s)
1 - - -
Gene 1
pLuxR
pLuxI
2 - -
C
B
Lux R
Gene 2
SgrS
tet R
X
LuxI
pRhiR
pRhlI
3 - -
E
A
RhlR
Gene 3
SgrS
tet R
Y
RhlI
?
?
4 - -
F
D
?
Gene 4
SgrS
tet R
Z
?
AB
AB
5 -
Gene 5
SgrS
Spot42
CD
CD
6 -
Gene 6
SgrS
Spot42
EF
EF
7 -
Gene 7
SgrS
Spot42
XYZ
XYZ
8
Spot42
Gene 8
6
Small RNAs in E. coli

• Were 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 Escherichiacoli small RNAs
  in vivo. Nucleic Acids Res. 200735(3)1018-37.
  Epub 2007 Jan 30.PubMed PMID 17264113 PubMed
  Central PMCID PMC1807950.

7
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-(3-Oxohexanoyl)-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.

8
Hybrid Systems

1. AB B2H1AB2H1B
2. CD B2H2AB2H2B
3. EF B2H3AB2H3B
4. XYZ B3H1AB3H1BB3H1C

Schematic of a 3-Hybrid System
Schematic of a 2-Hybrid System
9
Hybrid Systems Promoters
10 bp

• B2H1AB2H1B
• B2H2AB2H2B
• B2H3AB2H3B
• B3H1AB3H1BB3H1C

63 bp
Zif269BS
P(wk) weak Lac Promoter from E coli
10 bp
63 bp
TATA zifvar
P(wk) weak Lac Promoter from E coli
10 bp
P53zifvar
63 bp
P(wk) weak Lac Promoter from E coli
10 bp
OL2
62 bp
P(wk) weak Lac Promoter from E coli
10
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B2H1
Ala-Ala-Ala Linker
257
248
1
278
B2H1A
E. Coli RNA Polymerase Subunit A (residues 1-248)
Yeast Gal4 protein (residues 58-92)
AAAPVRTG Linker
113
89
1
207
B2H1B
Yeast Gal 11P (residues 263-352) N341V mutation
Zif 268 (residues 327-421)
13
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14
(No Transcript)
15
B2H2
Ala-Ala-Ala Linker
257
248
1
823
B2H2A
E. Coli RNA Polymerase Subunit A (residues 1-248)
GacS (residues 253-819)
AAAPVRTG Linker
113
89
1
B2H2B
GacA
TFIIB (DBD)
16
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B2H3
Ala-Ala-Ala Linker
257
248
1
B2H3A
E. Coli RNA Polymerase Subunit A (residues 1-248)
MavT
AAAPVRTG Linker
86
62
1
B2H3B
P53 (DBD)
MavU (residues 1-62)
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20
B3H1
B3H1A
FtsB
B3H1B
FtsL
B3H1C
CI (DBD)
FtsW
21

• Pros

• Cons

• It would be really cool and have lots
  interesting, novel aspects like the sRNA, hybrid
  systems, and autoinducer systems.
• A quick reaction time.
• Wed be introducing the hybrid system as a logic
  gate.
• With sRNAs and hybrid system, you can create any
  comination of gates.

• 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.

22
Questions?!?!?

• Time
• How much time would it take to make the 24?
• How much time would it then take to complete the
  38?
• How practical is it to assume that well be able
  to recreate the literature
• sRNAs
• Hybrid Systems
• Autoinducers
• Are acyl-ACP and SAM naturally produced in E.
  coli?

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The End
24
Gene of Interest
PA1, PAL1, PA2
A 0 0 0
tet
Gene A
TET
Luxpr
pLaxCl
B 1 0 0
C
B
Lux R
Gene B
SgrS
Spot42
tet R
pLAS
pLaS
C 0 1 0
E
A
LAS R
Gene C
GcvB
OxyS
tet R
pLUX
pLux
D 0 0 1
F
D
Lux Q
Gene D
RhyB
MicC
tet R
V
AB
AB
AB
E 1 1 0
Gene E
Spot 42
MicA
GcvB
CD
CD
CD
F 1 0 1
Gene F
SgrS
MicA
RhyB
EF
EF
EF
G 0 1 1
Gene G
MicC
MicA
OxyS
VW
VW
AB
H 1 1 1
W
MicA
Gene H
25
Gene of Interest
System using 2 small RNAs
PA1, PAL1, PA2
A 0 0 0
tet
Gene A
TET
Luxpr
pLaxCl
B 1 0 0
C
B
Lux R
Gene B
Spot 42
tet R
pLAS
pLaS
C 0 1 0
E
A
LAS R
Gene C
Spot 42
tet R
pLUX
pLux
D 0 0 1
F
D
Lux Q
Gene D
Spot 42
tet R
V
AB
AB
E 1 1 0
Gene E
Spot 42
MicA
CD
CD
F 1 0 1
Gene F
Spot 42
MicA
EF
EF
G 0 1 1
Gene G
Spot 42
MicA
VW
AB
H 1 1 1
W
Gene H
26
Gene of Interest
Same deal with a 3 Hybrid System
PA1, PAL1, PA2
A 0 0 0
tet
Gene A
TET
Luxpr
pLaxCl
B 1 0 0
C
B
Lux R
Gene B
Spot 42
tet R
X
pLAS
pLaS
C 0 1 0
E
A
LAS R
Gene C
Spot 42
tet R
Y
pLUX
pLux
D 0 0 1
F
D
Lux Q
Gene D
Spot 42
tet R
Z
AB
AB
E 1 1 0
Gene E
Spot 42
MicA
CD
CD
F 1 0 1
Gene F
Spot 42
MicA
EF
EF
G 0 1 1
Gene G
Spot 42
MicA
XYZ
XYZ
H 1 1 1
MicA
Gene H
27
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 Escherichiacoli small RNAs in
  vivo. Nucleic Acids Res. 200735(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.pro
  xy2.library.uiuc.edu/science?_obArticleURL_udiB
  6WSN-4VNHRSC-B_user571676_coverDate022F202F2
  009_rdoc10_fmthigh_origbrowse_srchdoc-info
  (23toc237051232009239986399952393309123FLA2
  3display23Volume)_cdi7051_sortd_docanchor_
  ct22_acctC000029040_version1_urlVersion0_u
  serid571676md5db7004c2567e64a29f9508281abc76ac

28
Category Number Examples Size (nt) Mechanism/role Regulators/comments References
Hfq-binding, antisense 22 DsrA 85 Stimulates rpoS Inhibits hns Low temp., LeuO 58, 75
RprA 105 Stimulates rpoS RcsC/B phosphorelay 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
Antisense 3 RyeA/SraC 275 Anti-RyeB Unknown 100
29
sRNAs that were going to use
SgrS http//www.ncbi.nlm.nih.gov.proxy2.library.uiuc.edu/pubmed/18042713?ordinalpos1itoolEntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum
Spot42 http//search.ebscohost.com/login.aspx?directtruedbaphAN14891380siteehost-live (sign into the U of I library)
OxyS http//www.sciencedirect.com.proxy2.library.uiuc.edu/science?_obMImg_imagekeyB6WSN-419B8BT-7-C_cdi7051_user571676_origbrowse_coverDate072F112F1997_sk999099998viewcwchpdGLzVzz-zSkzkmd5d29bac7ead27fdc1b702edb28fcadd11ie/sdarticle.pdf http//www.ncbi.nlm.nih.gov/pubmed/9230301?logactivity
GcvB http//web.ebscohost.com.proxy2.library.uiuc.edu/ehost/pdf?vid2hid105sida661093e-1c90-4ffa-be77-2b4e7f4b00b540sessionmgr102dbhchAN6012035
MicC https//ms5.express.cites.uiuc.edu/wm/mail/fetch.html?urlid75b51be9449ffa2eb80ed26874c1e5b44urlhttp3A2F2Fwww.pubmedcentral.nih.gov.proxy2.library.uiuc.edu2Farticlerender.fcgi3Ftool3Dpubmed26pubmedid3D15466019
RyhB http//www.pnas.org/content/99/7/4620.full
MicA Access the most recent version at doi10.1101/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
30
MicA secondarystructure and binding
31
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.
32
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.
33
OxyS
It negatively controls oxidative stress response
within the cell.
34
Spot 42
35
SgrS
36
GcvB
37
Quantification of Lux system

• http//www.pubmedcentral.nih.gov/picrender.fcgi?ar
  tid176701blobtypepdf (This is not as
  applicable)
• http//www.sciencedirect.com.proxy2.library.uiuc.e
  du/science?_obArticleURL_udiB6WBK4PRHJ6K2_user
  571676_rdoc1_fmt_origsearch_sortdviewc
  _acctC000029040_version1_urlVersion0_userid
  571676md5ea6566137620b30f76b459cf252ad23a
• (Log into the U of I library online database
  first)

38
Efficiency of Autoinducers
They used 3-oxo-hexanoyl-homoserine lactone (OHHL)
39
Kinetics of Autoinducers
Were using a non-feedback system, so look at the
triangles and the green.
40
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
41
Hybrid Systems

1. AB B2H1AB2H1B
2. CD B2H2AB2H2B
3. EF B2H3AB2H3B
4. VW B2H4AB2H4B
5. XYZ B3H1AB3H1BB3H1C

Schematic of a 2-Hybrid System
1
2
a
RNA Pol
Zif
Promoter
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Yeast 2-Hybrid System
1
2
a
RNA Pol
Zif
P(wk) weak Lac Promoter from E coli
10 bp
10 bp
63 bp
10 bp
P(wk) weak Lac Promoter from E coli
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B2H1A aGal4 protein
Ala-Ala-Ala Linker
257
248
1
278
E. Coli RNA Polymerase Subunit A (residues 1-248)
Yeast Gal4 protein (residues 58-92)
On pACYC184 derived pACL- aGal4 protein ? 1
PTG-inducible 1pp/lacUr5
46
B2H1B Gal 11P Zif 123
AAAPVRTG Linker
113
89
1
207
Yeast Gal 11P (residues 263-352) N341V mutation
Zif 268 (residues 327-421)
On pBR-GP-2123 ? Phagemid
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