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Team:UANL Mty-Mexico/Contributions/Photochassis
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Revision as of 23:27, 26 September 2011
Since light induction is becoming increasingly used in synthetic biology, we decided to create a built-in light induction system in E. coli through chromosome insertion. Avoiding the need of any extra-chromosomal DNA when light-inducing gene expression offers several advantages to the researcher. We therefore propose these modified E. coli strains as photo-chassis that could make useful tools in the field.
Chromosome integration will be performed through a two-step method for the insertion of large DNA fragments into any desired location in the E. coli chromosome, designed by Kuhlman and Cox[1]. Light induction genes will be obtained from plasmids constructed by Dr. Tabor et al.[2].
Ideally, three photo-chassis will be built: the first enabling green light induction, the second enabling red-light induction, and the third enabling both green and red lights induction in the same cell. A common chromophore is shared by the three strains. All genes and biobricks used for this purpose are listed at the bottom of the page.
Red Photo-chassis. Genes ho1 and pcyA are responsible for the chromophore synthesis. Cph8 codes for the chimaeric red-light receptor[3]. These three genes are constitutively expressed. Mnt repressor is expressed from pOmpC promoter, which stops being induced in presence of red-light. It is therefore used as a NOT-gate to regulate expression from pMnt (see Circuit Cell One).
Red photo-chassis.
Green Light Photo-chassis. Genes ho1 and pcyA are responsible for the chromophore synthesis. CcaS and ccaR code for the two-component green-light receptor. Absorption of green light increases the rate of CcaS autophosphorylation, phosphotransfer to CcaR, and transcription from the promoter of the cpcG2 promoter[2]. All four genes are constitutively expressed.
Green photo-chassis.
Red and Green light Photo-chassis. Assembles both constructions above with only one chromophore synthesis complex.
Red/Green photo-chassis.
|
Red photocassette |
||
|
Part |
Size |
Source |
|
pConst. + RBS |
58 bp |
|
|
Double terminator (TT) |
129 bp |
|
|
pOmpC |
108 bp |
|
|
mnt |
288 bp |
|
|
ho1 |
723 bp |
Tabor et al |
|
pcyA |
747 bp |
Tabor et al |
|
cph8 |
2235 bp |
Tabor et al |
|
Green photocassette |
||
|
Part |
Size |
Source |
|
pConst. + RBS |
58 bp |
|
|
Double terminator (TT) |
129 bp |
|
|
ho1 |
723 bp |
Tabor et al |
|
pcyA |
747 bp |
Tabor et al |
|
ccaS |
2262 bp |
Tabor et al |
|
ccaR |
705 bp |
Tabor et al |
- Kuhlman TE and Cox EC (2010) Site-specific chromosomal integration of large synthetic constructs Nucleic Acids Res 38:e92.
- Tabor JJ, Levskaya A, Voigt CA (2010) Multichromatic Control of Gene Expression in Escherichia coli. J Mol Biol 405:315-324.
- Levskaya A et al.(2005) Engineering Escherichia coli to see light. Nature 438:24.
