Team:XHD-Wuhan-B-China/Improve

Improvement

PcpcG2-68 is a truncated promoter from PcpcG2-172.

The original ccaS/ccaR/cpcG2 cassette was amplified from the genome of Synechocystis PCC6803. The open reading frame of the output gene cpcG2 was then seamlessly replaced with lacZ [1]. The light-controlled promoter is then named PcpcG2-238 by Tabor's study, in which they found a constitutive promoter through BPROM prediction that contributes to leakiness and low dynamic range. They truncated PcpcG2-238 to create PcpcG2-172 [2]. We hypothesized that the upstream sequence of PcpcG2-172 contributes little to CcaR~P binding, therefore we decided to truncate PcpcG2-172 into PcpcG2-68 to reduce the length of PcpcG2 promoter.

We tested PcpcG2-172 and PcpcG2-68 under the regulation of a series of CcaRs with different RBS strength.

We constructed a series of circuits containing combinations of J61-RBSs and PcpcG2-68/172. A total of 14 pSC101 plasmids were co-transformed with p15A plasmid respectively into MG1655-△EnvZ strain. These strains were cultivated under red or green light for 5 hours using a 24-well plate adapted device, then tested for eGFP expression level by a plate reader.

The result reveals that in most cases, PcpcG2-68 is slightly weaker than PcpcG2-172, while exhibiting similar fold-change pattern to PcpcG2-172. To be noticed that after changing weaker RBSs, we found two combinations both have a higher fold-change than the original version of J61100. This result conforms with our model prediction: between the expression level of J23109-J61100 and J23117-J61100 there is a peak of fold-change yet to be achieved for both 68 and 172. Please view https://2021.igem.org/Team:XHD-Wuhan-B-China/Model for more details.

In conclusion, the truncated promoter PcpcG2-68 has the similar pattern under the control of green/red light as PcpcG2-172, but is shorter and more convenient to construct. For example, when constructing an array of sgRNAs, PcpcG2-68 has a big advantage over PcpcG2-172: reducing the volume of the circuit and cheaper to synthesis.

References
[1]. Tabor JJ, Levskaya A, Voigt CA. Multichromatic control of gene expression in Escherichia coli. J Mol Biol. 2011 Jan 14;405(2):315-24.
[2]. Schmidl SR, Sheth RU, Wu A, Tabor JJ. Refactoring and optimization of light-switchable Escherichia coli two-component systems. ACS Synth Biol. 2014 Nov 21;3(11):820-31.