Team:UANL Mty-Mexico/Contributions/Photochassis

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. Jeff J. Tabor[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.

1. Kuhlman TE, Cox EC (2010) Site-specific chromosomal integration of large synthetic constructs Nucleic Acids Res38:e92.
2. Tabor JJ, Levskaya A, Voigt CA (2010) Multichromatic Control of Gene Expression in Escherichia coli. J Mol Biol 405:315-324.
3. Levskaya A Levskaya A, Chevalier AA, Tabor JJ, Simpson ZB, Lavery LA, Levy M, Davidson EA, Scouras A, Ellington AD, Marcotte EM, Christopher AV(2005) Engineering Escherichia coli to see light. Nature 438:441-442.