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).In this way, constructions will look like this:
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 UANLBricksConstruction by cloning
One of the alternatives for constructing the photocassette is by cloning, as they were classic BioBricks, like shown in the next figure:
As its referred in the image above, this plan consisted on:
- Extraction of CDS corresponding to genes for photoreceptor system by PCR.
- 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.
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 below:
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:
- Cutting PCR products of CDS and terminator for ligation.
- 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.
- Constructions of CDS-terminator are joined to promoter by cutting, ligating and PCR.
- 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 planned 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.Primer design
Primers were designed 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:
|CDS||Theoric Ta||DNA template||Expected amplified product|
|ccaR||59 ºC||pJT122||768 bp|
|ccaS||58.9 ºC||pJT122||2311 bp|
|cph8||61 ºC||pCph8||2299 bp|
|ho1||61.6 ºC||pPLPCB(S)||772 bp|
|pcyA||59.3 ºC||pPLPCB(S)||806 bp|
- 94ºC - 30 sec
- 30 cicles of:
- 94ºC - 30 sec
- 54-64ºC - 30 sec
- 72ºC - 30 sec
- 72ºC - 5 min
|Gene||CDS extraction||Joined to terminator||Joint to promoter||Joint to promoter and terminator||Cloned into pCR2.1-TOPO|