Difference between revisions of "Team:KU Leuven/Research/Idea"
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Both cells are transfected with only one plasmid each (Figure 1). Both plasmids contain a temperature sensitive cI repressor protein, which is constitutively expressed. This repressor is only able to bind to the lambda promoter at temperatures below a certain threshold (between 30°C and 42°C). At elevated temperatures, cI is unable to bind to the promoter region enabeling RNA polymerase to start transcription. | Both cells are transfected with only one plasmid each (Figure 1). Both plasmids contain a temperature sensitive cI repressor protein, which is constitutively expressed. This repressor is only able to bind to the lambda promoter at temperatures below a certain threshold (between 30°C and 42°C). At elevated temperatures, cI is unable to bind to the promoter region enabeling RNA polymerase to start transcription. | ||
<sup><a href="#Villaverde1993">[6]</a></sup> | <sup><a href="#Villaverde1993">[6]</a></sup> |
Revision as of 12:17, 10 August 2015
![](https://static.igem.org/mediawiki/2015/b/b7/KU_Leuven_Researchbanner.jpg)
Idea
Intrigued by the patterns occuring in nature, we started our research to design possible interaction schemes and genetic circuits. Looking at specialised literature, we were able to find some interesting papers concerning the formation of patterns using
Escherichia coli
(E. coli). For example, Basu et al. created ring-like patterns based on chemical gradients of an acyl-homoserine lactone (AHL) signal that is synthesized by ‘sender’ cells. In receiver cells, designed genetic networks respond to differences in AHL concentrations
[1].
Liu et al. created periodic stripes of high and low cell densities of E. coli, by inhibiting cell motility when cell density was high
[2].
Combining our own innovative and altered chemotaxis intercellular relationship with basic principles from both papers allowed us to design our own circuit which will be elucidated in the following paragraphs.
Two different cell types, called A and B, interact and create patterns. In order to achieve the desired behavior, the cells used in our experiments are derived from K12
Escherichia coli
strains and have specific knockouts. Cell type A has a deletion of
tar and
tsr
, whereas in cell type B both
tar and
cheZ are knocked out. Tar and Tsr are two major chemotactic receptors, guiding the cells towards nutrients. When a certain small molecule binds one of these receptors, a signalling cascade is initiated. In our specific case cell’s A deletion of both
tsr and
tar causes them to be insensitive to leucine (as a repellent and attractant). Cell B on the other hand, who has lost only Tar is repelled by leucine.
[3]
Upon recognition of the small molecule (in this case leucine) the receptor transduces the signal through a set of Che proteins. This group of methylesterases and phosphatases regulates the the flagellar rotation (figure 1). In E. coli, the motors turn counterclockwise (CCW) in their default state, allowing the several filaments on a cell to join together in a bundle and propel the cell smoothly forward. The interaction of phospho-CheY and a motorprotein causes the motors to switch to clockwise (CW) rotation, inducing dissociation of the filament bundle and tumbling of the cell.
[4]
To regulate CheY activity, a protein called CheZ removes the phosphate group from it and inhibits its activity. Cells lacking this protein are not able to swim and will tumble excessively and incessantly
[5].
![](https://static.igem.org/mediawiki/2015/d/d6/KU_Leuven_plasmidB.png)
![](https://static.igem.org/mediawiki/2015/b/b7/KU_Leuven_Researchbanner.jpg)
References
[1] Subhayu Basu, Yoram Gerchman, Cynthia H. Collins, Frances H. Arnold & Ron Weiss “A synthetic multicellular system for programmed pattern formation” Nature 434 (2005): 1130-1134.
[2] Chenli Liu, Xiongfei Fu, Lizhong Liu, Xiaojing Ren, Carlos K.L. Chau, Sihong Li, Lu Xiang, Hualing Zeng, Guanhua Chen, Lei-Han Tang, Peter Lenz, Xiaodong Cui, Wei Huang, Terence Hwa, Jian-Dong Huang” Sequential Establishment of Stripe Patterns in an Expanding Cell Population” Science 334 no. 6053 (2011) : 238-241.
[3] Khan, S., and D. R. Trentham. “Biphasic Excitation by Leucine in Escherichia Coli Chemotaxis.” Journal of Bacteriology 186, no. 2 (2004): 588-592.
[4] Sarkar MK, Paul K, Blair D , “Chemotaxis signaling protein CheY binds to the rotor protein FliN to control the direction of flagellar rotation in Escherichia coli.” PNAS 107, no. 20 (2010): 9370-9375.
[5] Kuo, Scot C., and D. E. Koshland. “Roles of cheY and cheZ Gene Products in Controlling Flagellar Rotation in Bacterial Chemotaxis of Escherichia Coli.” Journal of Bacteriology 169, no. 3 (1987): 1307–14.
[6] Villaverde, A., A. Benito, E. Viaplana, and R. Cubarsi. “Fine Regulation of cI857-Controlled Gene Expression in Continuous Culture of Recombinant Escherichia Coli by Temperature.” Applied and Environmental Microbiology 59, no. 10 (1993): 3485–87.
[7] Clay Fuqua , Matthew R. Parsek , and E. Peter Greenberg “Regulation of Gene Expression by Cell-to-Cell Communication: Acyl-Homoserine Lactone Quorum Sensing” , Annu. Rev. Genet. (2001) 35:439–68
[8] Ulett, G. C. “Antigen-43-Mediated Autoaggregation Impairs Motility in Escherichia Coli.” Microbiology 152, no. 7 (2006): 2101–2110.
[9] James L. Cox, Betty J. Cox , Vincenzo Fidanza , David H. Calhoun “The complete nucleotide sequence of the iZvGMEDA cluster of Escherichia coli K-12” Gene 56, Issues 2–3 (1987): 185–198.
[10] Wittman, V., H. C. Lin, and H. C. Wong. “Functional Domains of the Penicillinase Repressor of Bacillus Licheniformis.” Journal of Bacteriology 175, no. 22 (1993): 7383–7390.