Part:BBa_K4868001:Design
PaqCI cloning site with Fluoride detection insertion sequence
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 941
Illegal NheI site found at 964 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1786
Illegal XhoI site found at 1486
Illegal XhoI site found at 1795 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 750
- 1000COMPATIBLE WITH RFC[1000]
Design
For the design of this part, several things have been taken into consideration. First of all, RFP has been codon optimized for E. coli to improve the expression efficiency of RFP. Regarding the promoters, the T7 promoter was specifically selected, since it is a high efficiency promoter, and our goal is to express a large quantity of our enzymes or any other defluorination enzyme. The riboswitch part of the insertion piece, however, has a constitutively active promotor, since the transcription should happen constitutively, but the translation, and thereby the expression of sfGFP, will be dependent in the presence of fluoride. The overhangs/overlaps for the Gibson assembly were designed by adding the 30 bp of each end of the linearized plasmid to the ends of the insertion piece and adding the overlaps in the middle of the insertion piece. This makes the part easy to adjust for insertion into other plasmids with the T7 expression system, by simply taking the last 30 bp of each end of the linearized plasmid of choice and adding them to the ends of the insertion sequence. In the PaqCI insertion site we decided to add RFP as a placeholder/selection marker. This is both convenient, because it indicates whether the plasmid has been assembled and transformed after Gibson assembly, by being able to observe expression of RFP under the induction of IPTG. Furthermore, it is also an indicator of successful Golden Gate cloning, since RFP is replaced by the protein of choice. This means that if colonies are fluorescing red after transformation of the Golden Gate assembly, the insertion of the enzyme wasn’t successful, but if they are not expressing RFP under the induction of IPTG it means that the enzymes most likely were inserted.