Difference between revisions of "Part:BBa K1385000"

Latest revision as of 16:43, 16 October 2014

CPCG2 promoter -> TetR

This part incorporates the CpcG2 promoter from Jeff Tabor's 2010 paper "Multichromatic Control of Gene Expression in Escherichia coli" and makes it an inverter. This light regulated promoter expresses TetR in the presence of light. This part should be used in conjunction with https://parts.igem.org/Part:BBa_K1017726 to activate the light regulated plasmid, and a reporter plasmid such as https://parts.igem.org/Part:BBa_J70311 which is a pTet promoter a reporter protein EYFP

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 my 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 the system in

We used this part in our light repressor system with Ptet driving EYFP. The cpcG2 promoter was leakier than expected, but when induced with ATC we could clearly see there is a difference between light and dark systems.

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_K1385000 is represented by PBJ with and without ATC.

As you can see, there is a notable difference with and without the chromophore with and without aTc. The promoter is leakier than expected. As you can see, very little fluorescence is occurring in the light, which is what we want. When we bind up the TetR though, fluorescence is still pretty low, which means we need to use more aTc to bind the system in order to get fluorescence back up to positive control levels (1).

Design Notes

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* found available in Moon Lab, at Washington University in St. Louis

References

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

Sequence and Features

Functional Parameters