Part:BBa_K4212046
SDP3
L1
Introduction to this part
This part contains 0A spacer, optimal RBS, Cas9 CDS, and B0015 terminator.
Our Experiment Design
Cas9 is a large protein, consisting of 1368 residues ( ≈4.1kb). Its synthesis from IDT as a single linear fragment was ruled out due to the complexity of the build. For this reason, we once again relied on customized overhangs which split the protein CDS’s in half to allow for a directional and scarless assembly. In particular, these would come together and reconstitute the CDS in a L0 vector (STK001). Then, by harnessing the destination backbone 1C and 0A spacer, Cas9 would be paired with the appropriate regulatory elements (RBS and terminator) to allow for its translation. Indeed, Cas9’s construction in 1C lacks a promoter as the transcriptional levels are regulated upstream by PsspB (1B vector).Notably, cas9 was the largest protein we attempted to clone across Sporadicate. As soon as linear fragments were obtained and resuspended at 50fmol/μL in water, we tested two different molar ratios of vector to insert (1:5 and 1:3) in L0 Golden Gate assemblies. This choice is motivated by the attempt to drive assembly of Cas9 in the destination backbone (1C) by enhancing the pool of CDS inserts in the reaction mix. However, following transformation in E. coli and recovery on LB Amp, only incorrect colonies were present at the 16h time point. Colony PCR cas9 level 0Considering results obtained at this stage, it became apparent that our initial strategy to clone Cas9 in STK001 was not optimal.
Our Experiment Result
Hence, we asked for guidance to our supervisors Mo and Joaquin, who recommended decreasing the molar ratio to 2:1 while increasing cutting times with BsmBI (from 3min to 5min) and the number of reaction cycles (from 30x to 60x). Indeed, due to the size of the final product, longer digestion phases coupled with more reaction cycles may have allowed for a more efficient golden gate cloning process.Subsequently, assemblies were set up and transformed in E. coli TOP10, to then being left to recover at 37C overnight. The following day, we noticed that no colonies grew, but we still decided to give the cells a larger time window to allow for growth given the large insert size. After the 20h mark from the initial transformation, a large number of colonies appeared. At that point, adhering to the cloning framework adopted across the summer, colonies were screened via colony PCR to shortlist positive clones. Positive hits were isolated and grown overnight to be miniprepped and sent for sequencing. Correct constructs were then cultivated for preparation of glycerol stocks. image (L0 with a lot of clones of pCas9)Having successfully assembled and secured Cas9’s CDS in the toolkit, it was now time to construct the final module of the D15-Cas9 operon (i.e., 1C). In particular, construct 1C starts with spacer 0A (replacing this time the promoter slot) and is followed by RBS optRBS, the full Cas9 CDS, and terminator B0015. To that end, a higher order assembly was performed utilizing pCas9 (L0) clone x, leading to colonies of 1C for screening. As usual, colony PCR, followed by Restriction digest analysis of the assembled plasmid was performed; correct assemblies were stored in 50% glycerol (1:1) at -80C.
References
[1] https://parts.igem.org/Part:BBa_B0015:Design
[2] https://doulix.com/biomodules/62PCZ1Z/
[3] Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J.A. & Charpentier, E. (2012) A programmable dual RNA-guided DNA endonuclease in adaptive bacterial immunity. Science (New York, N.Y.). 337 (6096), 816–821. doi:10.1126/science.1225829.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 3856
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 3856
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 3856
Illegal BglII site found at 1221 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 3856
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 3856
- 1000COMPATIBLE WITH RFC[1000]
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