Part:BBa_K4807023
rhtA insert
In order to achieve the overexpression of rhtA exporter and Human catalase, we employed pETDuet-1 (BBa_J100519)as our carry out vector. This vector features a ColE1/pMB1/pBR322/pUC origin of replication and it is ampicillin resistant.
pETDuet-1 is a high copy number plasmid that features two T7 promoters and two RBS sequences. However, it contains only one T7 terminator. As a result, each gene is expressed under its dedicated T7 promoter, but one of the inserted genes requires the inclusion of a T7 terminator (BBa_K4807002) for proper regulation. The rhtA insert (BBa_K4807023) is the one carrying the extra T7 terminator.
In order to assemble our constructs, two cloning steps needed to be performed. Firstly, the vector was linearized through digestion with the BglII restriction enzyme to allow the insertion of the CAT gene (BBa_K4807003). Subsequently, another digestion with SacI was necessary to clone the rhtA exporter gene (BBa_K1977000) into our vector.
In order to confirm our gene expression, an S-tag (BBa_K3630004) was incorporated in the catalase insert making it possible to detect through Western-Blotting. The rhtA exporter utilizes a 6xHis-tag which is already an integral part of the plasmid.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 334
Illegal AgeI site found at 460 - 1000COMPATIBLE WITH RFC[1000]
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