Team:UANL Mty-Mexico/Wet lab/Photocassette

Photocassette construction was planned to couple all the photorreceptor genes for each cell (as mentioned in the section Photochassis).

Construction Map of Cell One

Construction Map of Cell Two

Construction Map of RedGreen Cell

Note: Construction of photocassette was planned to be through UANLBricks, which refers to a different assembly standard. For more information about this, please refer to the section UANLBricks

One of the alternatives for constructing the photocassette is by cloning, as they were classic BioBricks, like shown in the next figure:

Cloning strategy for photocassette construction

As its referred in the image above, this plan consisted on:

1. Extraction of CDS corresponding to genes for photoreceptor system by PCR.
2. Conversion of classic BioBricks to UANLBricks by PCR, for parts:
• Constitutive promoter joined to ribosome binding site (BioBrick K081005), referred as pConst in the diagram
• Transcription terminator (BioBrick B0015) referred as TT in the diagram by PCR.
3. Cloning PCR products into pCR2.1-TOPO.
4. Joining pCont to each CDS and cloning them into pGEM-T vector previously recircularized.
5. Cloning constructions of pConst-CDS's into pTT vector (Transcription terminator into pGEM-T), generating complete genes.
6. Joining corresponding genes to couple into cassettes to each cell of HuBac community.

As primers were meant to be synthesized for each CDS and for promoter and terminator, there was an alternative for constructing the photocassette. It is shown bellow:

PCR&Cloning strategy for photocassette construction

PCR&Cloning strategy is composed of two sub-strategies. First, the non-BioBrick part begins as cloning strategy, with PCR of correspondent CDS, promoter and transcription terminator, but instead of cloning into pCR2.1-TOPO, it continued this way:

1. Cutting PCR products of CDS and terminator for ligation.
2. PCR directly from ligation with CDS forward primer (CDS-FWD) and terminator reverse primer (Term-RV). Only correct ligations of CDS to terminator would amplify with expected length.
3. Constructions of CDS-terminator are joined to promoter by cutting, ligating and PCR.
4. Once having complete genes (promoter-CDS-terminator), this contructions are cloned into pCR2.1-TOPO and then follow cloning strategy as mention in the last paragraph.

BioBrick part was planed to be performed through PCR of each part with VR-VF2 primers, allowing this to have significant amounts of insert compared to original vectors (avoiding BioBrick part purification). PCR products were digested with respective enzymes in order to introduce them into respective recipient vectors, after that, PCR with the same primers was made, assuming there would not be any undesired amplified product but the one which is into vector, because digestion of previous PCR products eliminated sites for amplifying. Construction of not-gate through this methodology would end with PCR amplification of complete composite part using specific primers for pOmpC and terminator, which would convert this BioBrick part to UANLBrick composite part.

Primers were design in order to extract exclusively the correspond CDS for each gene. Additional primers were design to convert classic BioBricks to UANLBricks in case of constitutive promoter joined to RBS, transcription terminator and not-gate composite part. List of primers is shown below:

PCRs were performed as shown in the next table:

PCR program:

1. 94ºC - 30 sec
2. 30 cicles of:
   1. 94ºC - 30 sec
   2. 54-64ºC - 30 sec
   3. 72ºC - 30 sec
3. 72ºC - 5 min

PCRs:

Extraction of CDS for photocassette construction: cph8, pcyA, ho1. Ta Gradient (Anneling temperature) was applied in order to obtain best unique amplified products.

Extraction of CDS for photocassette construction: ccaR and ccaS. Ta Gradient (Anneling temperature) was applied in order to obtain best unique amplified products.

CDS cloned into pCR2.1-TOPO. At least one of each CDS was successfully cloned into this plasmid.

Ligation of cph8 to transcriptio terminator through ligation-PCR. Control demonstrates ligation of transcription terminator.

Ligation of ccaR-terminator to promoter. Control demonstrates ligation of constitutive promoter.

Ligation of ccaS CDS to constitutive promoter (Lanes 7 to 9).

Constructed parts