biologic we’re going to use – the quorum system – small rnas – 2-hybrid systems (and a...

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