Difference between revisions of "Part:BBa K1385001"

Latest revision as of 16:20, 16 October 2014

Hybrid CpcG2 promoter -> TetR

This is BBa_K1385000 with a hybrid cpcG2 promoter created to make leaky/strong expression of tetR.

CpcG2 hybrid promoter: Maintains Upstream region of promoter with G-box, the binding site of CcaR to the promoter. A Phosphorylated CcaR binds to the G-box and transcribes the cpcg2 gene. G-box is a conserved sequence located -108 upstream of TSS

In this case, the g-box works as an activator for the light sensor mechanism.

Replaced downstream (including -35 and -10 regions) with TetR.

Usage and Biology

PcpcG2 is a promoter from the genome of Synechocystis sp. PCC6803. The promoter comes from Jeffrey Tabor's plasmid pJT122 plasmid, contains the entire region upstream of cpcG2 and downstream of ccaR (Tabor et al. 2011). PcpcG2 is regulated by the light-activation of ccaS/ccaR. The gene downstream to this promoter is transcribed upon activation by ccaS/ccaR, via green light.

In our experimental plasmid, the CcaR/CcaS/ CpcG2 promoter/ tetR/Tet promoter/EYFP are all on the same plasmid. I was not able to clone the entire plasmid since it is a modified version of Tabor's PJT122 and the CcaR/CcaS genes had illegal restriction sites, so I decided only to biobrick the promoter with tetR. CcaR/CcaS genes can be found on the registry, as well as tet promoters driving reporter proteins; in my case I decided to use EYFP.

The light activation system is as follows:

This promoter needs to be used in conjunction with the Phycocyanobilin (PCB) which converts Heme and PcyA and Ho1 into the chromophore (why you need to use it with https://parts.igem.org/Part:BBa_K1017726). It also needs the CcaR and CcaS genes in order to function. Therefore you need a part such as https://parts.igem.org/Part:BBa_K360051 to work.

When activated by light, this promoter transcribes an output gene, in this case TetR, creating an inverter mechanism for a gene driven by pTet. In our experimental plasmids we had this in conjunction with a pTet promoter driving a reporter protein.

Use

To Use this promoter, you need to co-transform with the Phycocyanobilin Plasmid such as BBa_K1017726. You also need the CcaR/CcaS part such as BBa_K360051. And a Ptet promoter driving your desired gene. Then you have to grow cultures in either green light (535nm) or broad spectrum light to produce high levels of TetR.

This is the plasmid we used it in

We used this part in our light repressor system with Ptet driving EYFP. The hybrid promoter was leakier than expected but provided greater dynamic fold change when both systems were induced with ATC. In red was when results differed from our predicted.

We ran an experiment where we grew a set of cultures overnight. We diluted them all to OD 0.1, grew for two hours and then induced half with 250 ng/mL of aTc, which binds up tetR, and the other half we just let it grow. We let the cultures grow for an additional 7 hours under broad spectrum light, spun them down, and resuspended in 200µl of 1x Phosphate Buffered Saline. We then measured the fluorescence values 96 well dark plates with the settings of Absorption = 600nm, Fluorescence gain = 90, Excitation: 485nm, Emission: 528nm.

The results we got were normalized to the positive control in three separate wells. How we calculated them was we took the average of the negative control, subtracted all of the values. Then divided them all by the average of the positive control. From there we found the average and standard deviation of our cultures. The Normalized Fluorescence as presented is Average +/- SD.

BBa_K1385001 is represented by HYB with and without ATC.

As you can see, in the light our promoter is functioning as expected, namely our system is completely turned off. Our numbers were even lower than the negative control, hence we displayed them as 0 for Hybrid 0 aTc. Even when we added a good amount of aTc, there is very little fluorescence, so there is ALOT of tetR around. We cannot conclude that the hybrid promoter is light induced but we know it is very leaky and did it's job. We never want any fluorescence when there is light around, otherwise it would be wasteful.

Design Notes

G-box, hybrid promoter ptrc1O The spacer region between TetR and cpcG2 can be modified in order to increase or decrease the RBS strength, as TetR start codon is a ways down from the end of the promoter. The region it is so far is because our primers when assembling the plasmid had troublesome secondary structures closer to the promoter and start codon, so we needed a spacer.

The reason we made it an inverter was because we wanted to turn off transcription in the light. We wanted transcription of our target gene to only be in the dark as our big picture is to use this in organisms that can both photosynthesize and fix nitrogen as separate processes. By separating the two into light/dark cycles, nitrogenase will be sheltered from high amounts of cellular oxygen that is a product of photosynthesis.

Source

cpcG2 promoter Genes from PJT122 (Tabor lab)
TetR from a plasmid Ptet-pp* available in Moon Lab, at Washington University in St. Louis
Trc1O from a plasmid pSL2264 available in Pakrasi Lab, at Washington University in St. Louis

References

Yuu Hirose et al (2008) "Cyanobacteriochrome CcaS is the green light receptor that induces the expression of phycobilisome linker protein." PNAS vol. 105: 9528–9533.

Tabor, J. J. et al.(2010), " Multichromatic Control of Gene Expression in Escherichia coli", J. Mol. Biol. , doi:10.1016/j.jmb.2010.10.038

Hirose, Narikawa, et al. "Cyanobacteriochrome CcaS regulates phycoerythrin accumulation in Nostoc punctiforme, a group II chromatic adapter" PNAS May 11, 2010 vol. 107 no. 19

Sequence and Features