iccdB Intelligent Control of Cell Density in Bacteria Xiamen - - PowerPoint PPT Presentation

Xiamen University 2011

iccdB

— — Intelligent Control of Cell Density in Bacteria

Our Team

• 9 Undergraduates
• 3 Advisors

Inspiration

Pu’er Tea Fermentation

iccdB

— — Intelligent Control of Cell Density in Bacteria A bacteria population-control device

LuxI Producer LuxR Producer CcdB Producer

Mechanism

IPTG LuxI AHL LuxR

AHL an acyl-[acp] s-adenosyl-L-methionine a holo-[acp] s-methyl-5'-thioadenosine + LuxR CcdB lacl+pL luxI lacl+pL luxR lux pR

Mechanism

lacZα-ccdB

IPTG LuxI AHL LuxR

LuxR CcdB lacl+pL luxI lacl+pL luxR lux pR

Mechanism

lacZα-ccdB

CcdB Gyrase

Mechanism

CcdB- Gyrase Complex

Mechanism

Performance

• Bacteria population control devices

24h 32h

Performance

A series of bacteria population control devices based on RBSes Mutation of promoter lux pR Influence of iccdB on expression

• f downstream genes

“Can we generate a series

• f devices that program

bacteria population maintain at different cell densities? ”

Performance

lux pR lacZα-ccdB CcdB RBS0.6 RBS0.3 RBS0.07 RBS1.0

Performance

• Different RBSes

14.7 6.92 4.45 2.53 1.68 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 BL21 iccdB0.07 iccdB0.3 iccdB0.6 iccdB1.0 Colony-Forming Units /mL-1 X10-8 Devices

Cell Growth Column Graph

Modeling

“What else? ”

Performance

Performance

• Mutations of lux pR

—— Expression efficiency

IPTG

luxI luxR gfp

EGFP lux pR AHL

lux pR gfp

ACCTGTAGG

lux pR gfp

ACCTGTAGG

lux pR gfp

ACCTGTAGG

• Mutations of lux pR

——Expression efficiency

gfp

AC T TAGG T C

gfp

ACCT TAGG C

gfp

AC TGTAGG T

• Mutations of lux pR

——Expression efficiency

56.54 100.00 11.89 6.02 0.00 20.00 40.00 60.00 80.00 100.00 120.00 lux pR-5 lux pR-3 lux pR lux pR-3/5 Efficiency /% Devices

Promoters Strength Relative to lux pR-3 (BBa_K658006)

lux pR - 3 lux pR - 5

lacZ α-ccdB EGFP CcdB lux pR-3

Performance

• Mutations of lux pR

—— iccdB-X

1.51 2.53 0.5 1 1.5 2 2.5 3 3.5 iccdB-3 iccdB0.6

Colony-Forming Units / mL-1 X10-8

Devices

Cell Growth Column Graph

iccdB0.6 lacl+pL gfp

Performance

• Influence of iccdB on

downstream genes

1602.4 1203.1 200 400 600 800 1000 1200 1400 1600 1800 GFP iccdB-GFP

Fluorescence Devices

Fluorescence of Different Devices

Induced by IPTG

luxI luxR ccdB coding

Future Plan

Designed and characterized a series of bacteria population-control devices. Improved the strength of promoter lux pR by site- directed mutagenesis. Characterized the efficiency of promters lux pR and its 3 mutants. Designed and characterized the device iccdB-GFP to test the influence of iccdB on downstream genes.

Summary

Scientific Achievement

Open lab day for kids Biosafety lecture

Human Practice

Open lab day for kids

• 20 kids from Yan Wu primary school
• Exhibition about microorganism
• Lecture about synthetic biology
• Basic experiment

8% 25% 10% 5% 27% 25% Arts Economics Lingusitic Chemistry Management Others

Biosafety lecture

45% 26% 18% 8% 3% Genetically modified foods Artificial microorganism Bio-leakage from laboratory Threat of Human Genome Project on Human rights Others Majors Top questions

Reference

[1] Kempner E, Hanson F. Aspects of light production by Photobacterium fischeri[J]. Journal of Bacteriology, 1968, 95(3): 975-979. [2] Nealson KH, Platt T, Hastings J. Cellular control of the synthesis and activity of the bacterial luminescent system[J]. Journal of Bacteriology, 1970, 104(1): 313-322. [3] Fuqua WC, Winan SC, Greenberg E. Quorum sensing in bacteria: the luxR-luxI family of cell density-responsive transcriptional regulators[J]. Journal of Bacteriology, 1994, 176(2): 269-275. [4] Eberhard A, Burlingame AL, Eberhard C, Kenyon GL, Nealson KH, Oppenheimer NJ. Structural identification of autoinducer of Photobacterium fischeri luciferase[J]. Biochemistry, 1981, 20(9): 2444-2449. [5] Engebrecht J, Nealson KH, Silverman M. Bacterial bioluminescence: isolation and genetic analysis of functions from Vibrio fischeri[J]. Cell, 1983. 32(3): 773-781. [6] Ruby EG, Nealson KH. Symbiotic association of Photobacterium fischeri with the marine luminous fish Monocentris japonica: a model of symbiosis based on bacterial studies[J]. The Biological Bulletin, 1976, 151(3): 574-586.

[7] Ruby EG, McFall-Ngai MJ. Oxygen-utilizing reactions and symbiotic colonization of the squid light organ by Vibrio fischeri[J]. Trends in Microbiology, 1999, 7(10): 414-420. [8] Kaplan HB, Greenberg E. Diffusion of autoinducer is involved in regulation of the Vibrio fischeri luminescence system[J]. Journal of Bacteriology, 1985, 163(3): 1210-1214. [9] http://www.che.caltech.edu/groups/fha/quorum.html [10] Baldwin T, Devine JH, Heckel, RC, Lin, JW, Shadel GS. The complete nucleotide sequence of the lux regulon of Vibrio fischeri and the luxABN region of Photobacterium leiognathi and the mechanism of control of bacterial bioluminescence[J]. Journal of Bioluminescence and Chemiluminescence, 1989, 4(1): 326-341. [11] Meighen EA. Enzymes and genes from the lux operons of bioluminescent bacteria[J]. Annual Reviews in Microbiology, 1988, 42(1): 151-176. [12] You L, Cox RS, Weiss R, Arnold FH. Programmed population control by cell-cell communication and regulated killing[J]. Nature, 2004, 428(6985): 868-871. [13] Kampranis SC, Howells AJ, Maxwell A. The interaction of DNA gyrase with the bacterialtoxin CcdB: evidence for the existence of two gyrase-CcdB complexes[J]. Journal of Molecular Biology, 1999, 293(3): 733-744.

Acknowledgements

Steve Jobs

Thank you