"
Team:UANL Mty-Mexico/Contributions/Photochassis
From 2011.igem.org
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 photochassis 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 (2010)[2].
Ideally, three photochassis 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 Photochassis. 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 photochassis.
Green Light Photochassis. 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 photochassis.
Red and Green light Photochassis. Assembles both constructions above with only one chromophore synthesis complex.
Red/Green photochassis.
| Red Photocassette | |||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|
Part |
Size |
Source |
|
pConst. + RBS |
58 bp |
K081005 |
|
Double terminator (TT) |
129 bp |
B0015 |
|
pOmpC |
108 bp |
R0082 |
|
mnt |
288 bp |
|
|
ho1 |
723 bp |
Tabor et al. (2010) |
|
pcyA |
747 bp |
Tabor et al. (2010) |
|
cph8 |
2235 bp |
Tabor et al. (2010) |
| Green Photocassette | ||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
|
Part |
Size |
Source |
|
pConst. + RBS |
58 bp |
K081005 |
|
Double terminator (TT) |
129 bp |
B0015 |
|
ho1 |
723 bp |
Tabor et al. (2010) |
|
pcyA |
747 bp |
Tabor et al. (2010) |
|
CcaS |
2262 bp |
Tabor et al. (2010) |
|
CcaR |
705 bp |
Tabor et al. (2010) |
- Kuhlman TE, 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, 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.
