sgRNA woth switchable crRNA sequence and RFP reporter Bba_K2654002 in pSB1C3
- Strong promoter J23119 Bba_R0010: This promoter is the strongest from a big combinatorial family : (J23100:J23119).
- Strong RBS B0034 : Strongest RBS from the family B0030:B0034
- Modified mRFP E1010 : Engineered mutant of the red fluorescent protein of Discosoma striata. We performed two silent mutations to avoid two forbidden restriction sites in the RFP gene: the codon 197-199 TAC was mutated in a TAT, and the codon 527-529 GAA was mutated into a GAG. After those mutations we verified that the RFP was still expressed and that there was no effect on fluorescence.
- TracRNA: We based the sequence of these gene on a review (Jiang et al., 2015), this sequence is considered mostly invariable.
- Terminator Bba_B0010.
This biobrick was submitted to become a basic part in the iGEM collection
LacI inducible promotor Bba_R0010
This LacI regulated promoter was used in our reporter plasmid to promote the experession of the GFP coding cassette (See Composite parts).
Where does this biobrick come from and how to use it:
The idea was to make a Biobrick where the variable region of the sgRNA could easily be modified by an assembly with two oligonucleotides. The next step would be to assemble multiple modified biobricks to obtain an multiplex of sgRNA’s in a cloning vector. Finally, this cassette can be integrated with another biobrick by the 3A assembly protocol .
BaeI is a 38 pb site : it cuts two times at the 3’ extremity leaving 5pb protuberances. We used this restrictiction site among other things, because it doesn’t cut in the conjugative plasmids we use (pSW23T and pK18mob). It requires the use of 2x33pb oligonucleotides as primers.
BaeI sites in Bba_K2654002:
Consensus oligo :
direct (5’-3’) : cNNNNNNNNNNNNNNNNNNNNgttttagAGCTA
(Attention N is the complementeray of the direct strand)
reverse (5’-3’) : ctaaaacRRRRRRRRRRRRRRRRRRRRgCTAGT
(Attention R is the complementary of the reverse strand)
Figure 2: Digestion of pUC19_K2654002 by BaeI
ex : Target RFP (Bba_J04450) :
direct: 5’ cACTGCTAGCTAGCTAGTCATgttttagAGCTA 3’
reverse: 5’ ctaaaacATGACTAGCTAGCTAGCAGTgCTAGT 3’
direct: 5’ ccatgcgtttcaaagttcgtagttttagAGCTA 3’
reverse: 5’ ctaaaactacgaactttgaaacgcatggCTAGT 3’
Sigma-Aldrich propose a protocol for annealing those nucleotides. It can be found here: Protocol for annealing oligonucleotides, Sigma-Aldrich
Jiang, Y., Chen, B., Duan, C., Sun, B., Yang, J., Yang, S., 2015. Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System. Appl. Environ. Microbiol. 81, 2506–2514. https://doi.org/10.1128/AEM.04023-14