First of all, we needed a system that could cause a change in its medium conductivity. An extrusion pump seemed to be the best scheme to achieve this. Once this was devised, we needed a mechanism to regulate the system. We decided to use a negative regulator because it's the only way to transcriptionally regulate the expression of a gene in a definitive way.
We had to be able to restart our system, so we could add a signal at anytime. This could be accomplished with an induction signal that disappears rapidly after its involvement. The need of a link between the inductor signal and the repressor, lead us to include a little regulation cascade. This cascade allows us to add new steps which might increase our system’s complexity.
The components selected to fulfill the system requirements are enlisted in the next table:
* All the references for this table are included at the end of the design section.
For the assembly of the devices, the oligos contain restriction sites that are compatible to each vector or subsequent part. The synthesized primers were designed to carry the following operators and promoters in order to introduce them in the devices.
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The upper primer of the RcnA contain the cI and RcnR promoters in the BBa_K119009 part.
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For the BBa_K119010/BBa_119011 the upper primer of AiiA contain the LacZ promoter and the modified LacZ promoter.
Device BBa_K119009: The extrusion pump.
The propose of this device is to manipulate the transcription of rcnA by an inhibitory signal while maintaining the natural regulation of rcnA through RcnR. To achieve this the device contains a CI dependent promoter, RcnR binding site and the RcnA extrusion pump inserted in the vector pBBR1MCS-5.
Devices BBa_K119010/BBa_K119011: The regulatory device
In order to control the RcnA activity this device includes the gene encoding LuxR under the regulation TetR constitutive promoter followed by cI, which will repress RcnA in the prescence of AHL:LuxR. The last component of the device is the gene encoding AiiA. In BBa_K119010 lacZ promoter is upstream of AiiA, while BBa_K119011 carries a mutated version of it. The plasmid carrying this device will be PRK415.
We intend to measure variations in resistivity in a medium with a bacteria culture. This is achieved using an electronic system.
First of all we need a dispositive capable of detecting small resistivity variations. To achieve this, a resistive array in a Wheatstone bridge configuration is implemented.
To process the signal a Digital-Analogical capture card with an USB communication interface will be used. This will allow analogical data acquisition and its transfer to a computer on a binary format.
References
1.-Koch, D., Nies, D.H., Grass G.”.(2006) "The RcnR (YohLM) system of Escherichia coli: A connection between nickel cobalt and iron homeostasis"
2.-Rodrigue A. Et al. (2005) "Identification of rcnA (yohM), a Nickel and Cobalt Resistance Gene in Esherichia coli"
3.-Kovach et al.(1994), "pBBR1MCS: a broad-host-range cloning vector".
4.-Parsek MR,(1999) Acyl homoserine-lactone quorum-sensing signal generation.Apr 13;96(8):4360-5.
5.-http://partsregistry.org/Part:BBa_I729006
6.-Whiteheada N.A., Barnada A.M.L., Slaterra H.(2001) "Quorum-sensing in Gram-negative bacteria" .
7.-Fuqua, W.C., Winans, S.C., Greenber, E.P.(2001).”Quorum sensing in bacteria: The LuxR-LuxI family of cell densisty-responsive transcriptional regulators”.
8.-Salmond, G.P.C., Bycroft, B.W., Stewart, G.S.A.B., Williams, P.(1995).”The bacterial 'enigma': Crackin the code of cell-cell communication”.
9.-Y. Dong and L. Zhang,(2005). “Quorum sensing and quorum-quenching enzymes”.
10.-Atsumi, S., Little, J.W.(2006). “A synthetic phage λ regulatory circuit”.
11.- Karzai, A.W.(2000)."The Ssra-SmpB system for protein tagging, directed degradation and ribosome rescue".
12.-Keiler, K.C.et al.(1996)."Role of a peptide-tagging system in degradation of proteins synthesized from damaged messenger RNA" .
13.-Malan, T. P., A. Kolb, H. Buc, and W. R. McClure (1984). Mechanism of CRP-cAMP activation of lac operon transcription initiation activation of the P1 promoter. J. Mol. Biol. 180:881–909.
14.-J. Togashi, K. Ueda and T. Namai,(2001). “Overwintering of Erwinia carotovora subsp. carotovora in diseased tissues in soil and its role as inoculum for soft rot of Chinese cabbage”
15.-Y. Dong and L. Zhang,(2005). “Quorum sensing and quorum-quenching enzymes”.
16.- (1998). N.T. Keen, S. Tamaki, D. Kobayashi, and D. Trollinger.
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