Team:LCG-UNAM-Mexico/Experiments/Design

System

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, you can click on it to see a larger version:

* All the references for this table are included at the end of the design section.

Primer design

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.

• The upper primer of the RcnA contain the cI and RcnR promoters in the BBa_K119009 part.
• For the BBa_K119010/BBa_119011 the upper primer of AiiA contain the LacZ promoter and the modified LacZ promoter.
  
  

Devices

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.

Note: Our final bioparts were send to the registry in the standar plasmid pSB1A2.

Sensing dispositive

We intend to measure variations in resistivity in a bacteria culture which has been exposed to nickel . This is achieved through 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 an Analogical-Digital 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.

Detection of conductivity variations in the bacterial culture is achieved by introducing two platinum-covered chrome electrodes into the medium. Since high medium resistivity is expected, we use four resistances of 100 kΩ, one of them is variable.

Efflux and internalization parameters

In order to determine the nickel internalization and extrusion parameters a gradient of NiSO4 concentrations from 1x10-3 to 1X10-10 was tested. The growth inhibitory and the minimum RcnR inhibitory concentrations were included in the analysis as well.

Before to start with the measurements the electronic system’s sensitivity should be tested. In order to achieve this and get a reference signal which would let us eliminate noise, measurements where done with just LB, LB with NiSO4 and LB with cells.

Internalization

In order to determine the nickel internalization parameter liquid cultures of the YohM- strain will be needed at an O.D. (Optical density) 0.5 at a lambda of 600nm, where resistivity will be measured for different concentrations of NiSO4. YohM- strain was selected because as it lacks RcnA, so it will be internalizing nickel all the time. This may facilitate to get the wished parameter as the changes in the resistivity medium might be caused by the internalization pump alone.

Extrusion

The following steps were performed in order to prove the RcnA activity, and to get the enough data to calculate conductivity from the resistivity measurements. These data will be used to get the extrusion rate of RcnA by a relation between the conductivity and divalent ions concentration.

Further details on notebook.

Full sensing dispositive explanation here.

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"BioMetals 20 (5), pp. 759-771
2.-Rodrigue A. Et al. (2005) "Identification of rcnA (yohM), a Nickel and Cobalt Resistance Gene in Esherichia coli" Journal of Bacteriology 187 (8), pp. 2912-2916
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" . FEMS Microbiology Reviews 25 (4), pp. 365-404
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”. Annual Review of Microbiology 50, pp. 727-751
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”. Molecular Microbiology 16 (4), pp. 615-624
9.-Y. Dong and L. Zhang,(2005). “Quorum sensing and quorum-quenching enzymes”. Journal of Microbiology 43, pp. 101-109
10.-Atsumi, S., Little, J.W.(2006). “A synthetic phage λ regulatory circuit”. Proceedings of the National Academy of Sciences of the United States of America 103 (50), pp. 19045-19050
11.- Karzai, A.W.(2000)."The Ssra-SmpB system for protein tagging, directed degradation and ribosome rescue". Nature Structural Biology 7 (6), pp. 449-455
12.-Keiler, K.C.et al.(1996)."Role of a peptide-tagging system in degradation of proteins synthesized from damaged messenger RNA" . Science 271 (5251), pp. 990-993
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” J Gen Plant Pathology 67, pp. 45-50
15.-Y. Dong and L. Zhang,(2005). “Quorum sensing and quorum-quenching enzymes”. Journal of Microbiology 43, pp. 101-109
16.- (1998). N.T. Keen, S. Tamaki, D. Kobayashi, and D. Trollinger.