biologic we’re going to use – the quorum system – small rnas – 2-hybrid systems (and a...
TRANSCRIPT
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 Systems1. A+B B2H1A+B2H1B2. C+D B2H2A+B2H2B3. E+F B2H3A+B2H3B4. X+Y+Z B3H1A+B3H1B+B3H1C
Schematic of a 2-Hybrid SystemSchematic of a 3-Hybrid System
1 - - -
4 - - +
2 + - -
3 - + -
Gene 1
pLuxR
TET
CBLux R
pLuxI
Gene 2 SgrStet R
pRhiR
EA RhlR
pRhlI
Gene 3 SgrStet R
?
FD?
?
Gene 4SgrStet R
5 + + -
7 - + +
6 + - +
8 + + +
Z
AB
Gene 5
AB
SgrS
CD
Gene 6
CD
EF
Gene 7
EF
XYZ
Gene 8 Spot42
SgrS
SgrS
Spot42
Spot42
Spot42
Gene, Autoinducer(s)
XYZ
Y
XLuxI
RhlI
?
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-(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.
Hybrid Systems1. A+B B2H1A+B2H1B2. C+D B2H2A+B2H2B3. E+F B2H3A+B2H3B4. X+Y+Z B3H1A+B3H1B+B3H1C
Schematic of a 2-Hybrid SystemSchematic of a 3-Hybrid System
Hybrid Systems Promoters
P(wk); weak Lac Promoter from E coli
63 bp
10 bp
P(wk); weak Lac Promoter from E coli
63 bp
10 bp
P(wk); weak Lac Promoter from E coli
63 bp
10 bp
P(wk); weak Lac Promoter from E coli
62 bp
10 bp
Zif269BS
P53zifvar
TATA zifvar
OL2
B2H1A+B2H1B
B2H2A+B2H2B
B2H3A+B2H3B
B3H1A+B3H1B+B3H1C
B2H1
2572481 278
Ala-Ala-Ala Linker
E. Coli RNA Polymerase Subunit A (residues 1-248)
Yeast Gal4 protein (residues 58-92)
113891 207
AAAPVRTG Linker
Yeast Gal 11P (residues 263-352)*N341V mutation
Zif 268 (residues 327-421)
B2H1A
B2H1B
B2H2
2572481 823
Ala-Ala-Ala Linker
E. Coli RNA Polymerase Subunit A (residues 1-248)
GacS(residues 253-819)
113891
AAAPVRTG Linker
GacA TFIIB (DBD)
B2H2A
B2H2B
B2H3
2572481
Ala-Ala-Ala Linker
E. Coli RNA Polymerase Subunit A (residues 1-248)
MavT
86621
AAAPVRTG Linker
MavU (residues 1-62)
P53 (DBD)
B2H3A
B2H3B
B3H1
B3H1A
B3H1B
B3H1C
FtsL
FtsB
CI (DBD)FtsW
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
A 0 0 0
D 0 0 1
B 1 0 0
C 0 1 0
tet Gene A
pLaxCl
TET
CBLux R
Luxpr
Gene B SgrSSpot42tet R
pLaS
EALAS R
pLAS
Gene C GcvBOxyStet R
pLux
FDLux Q
pLUX
Gene D RhyBMicCtet R
E 1 1 0
G 0 1 1
F 1 0 1
H 1 1 1
V
AB
Gene E
AB
Spot 42
CD
Gene F
CD
EF
Gene G
EF
AB VW
Gene HW MicA
SgrS
MicC
MicA
MicA
MicA
PA1, PAL1, PA2
GcvB
RhyB
OxyS
AB
CD
EF
VW
Gene of Interest
A 0 0 0
D 0 0 1
B 1 0 0
C 0 1 0
tet Gene A
pLaxCl
TET
CBLux R
Luxpr
Gene B Spot 42tet R
pLaS
EALAS R
pLAS
Gene C Spot 42tet R
pLux
FDLux Q
pLUX
Gene D Spot 42tet R
E 1 1 0
G 0 1 1
F 1 0 1
H 1 1 1
V
AB
Gene E
AB
Spot 42
CD
Gene F
CD
EF
Gene G
EF
AB VW
Gene HW
Spot 42
Spot 42
MicA
MicA
MicA
PA1, PAL1, PA2
System using 2 small RNAs
Gene of Interest
A 0 0 0
D 0 0 1
B 1 0 0
C 0 1 0
tet Gene A
pLaxCl
TET
CBLux R
Luxpr
Gene B Spot 42tet R
pLaS
EALAS R
pLAS
Gene C Spot 42tet R
pLux
FDLux Q
pLUX
Gene D Spot 42tet R
E 1 1 0
G 0 1 1
F 1 0 1
H 1 1 1
Z
AB
Gene E
AB
Spot 42
CD
Gene F
CD
EF
Gene G
EF
XYZ
Gene H MicA
Spot 42
Spot 42
MicA
MicA
MicA
PA1, PAL1, PA2
XYZ
Same deal with a 3 Hybrid System
Y
X
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. 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-4VNHRSC-
B&_user=571676&_coverDate=02%2F20%2F2009&_rdoc=10&_fmt=high&_orig=browse&_srch=doc-info(%23toc%237051%232009%23998639995%23933091%23FLA%23display%23Volume)&_cdi=7051&_sort=d&_docanchor=&_ct=22&_acct=C000029040&_version=1&_urlVersion=0&_userid=571676&md5=db7004c2567e64a29f9508281abc76ac
Category Number Examples Size (nt) Mechanism/role
Regulators/comments
References
Hfq-binding, antisense
22 DsrA 85 Stimulates rpoSInhibits 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
sRNAs that we’re going to use:SgrS http://www.ncbi.nlm.nih.gov.proxy2.library.uiuc.edu/pubmed/18042713?
ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum
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=B6WSN-419B8BT-7-C&_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=a661093e-1c90-4ffa-be77-2b4e7f4b00b5%40sessionmgr102#db=hch&AN=6012035
MicC https://ms5.express.cites.uiuc.edu/wm/mail/fetch.html?urlid=75b51be9449ffa2eb80ed26874c1e5b44&url=http%3A%2F%2Fwww.pubmedcentral.nih.gov.proxy2.library.uiuc.edu%2Farticlerender.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.354405Genes Dev. 2005 19: 2355-2366Klas I. Udekwu, Fabien Darfeuille, Jörg Vogel, et al.antisense RNAHfq-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.
OxySIt 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=B6WBK4PRHJ6K2&_user=571676&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000029040&_version=1&_urlVersion=0&_userid=571676&md5=ea6566137620b30f76b459cf252ad23a– (Log into the U of I library online database first)
Efficiency of Autoinducers
They used 3-oxo-hexanoyl-homoserine 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 Systems1. AB B2H1A+B2H1B2. CD B2H2A+B2H2B3. EF B2H3A+B2H3B4. VW B2H4A+B2H4B5. XYZ B3H1A+B3H1B+B3H1C
Promoter
RNA Polα
1
Zif
2
Schematic of a 2-Hybrid System
P(wk); weak Lac Promoter from E coli
RNA Polα
1
Zif
2
P(wk); weak Lac Promoter from E coli
63 bp
10 bp
10 bp
10 bp
Yeast 2-Hybrid System
B2H1A; αGal4 protein
2572481 278
Ala-Ala-Ala Linker
E. Coli RNA Polymerase Subunit A (residues 1-248)
Yeast Gal4 protein (residues 58-92)
On pACYC184 – derived pACL- αGal4 protein 1 PTG-inducible 1pp/lacUr5
B2H1B; Gal 11P – Zif 123
113891 207
AAAPVRTG Linker
Yeast Gal 11P (residues 263-352)*N341V mutation
Zif 268 (residues 327-421)
On pBR-GP-2123 Phagemid
