Part:BBa_K345667:Design
Lac regulated insertable promoter
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Making a Controlled T7 Expression Vector
The RMIT2010 team is interested in producing a protein expression vector for producing peptides fused with Taq. Arguably the best protein expression systems are the pET vectors. These vector systems use a T7 promoter regulated by the Lac Operon elements.
This part has the -35 and -10 regions mutated to restriction sites to allow the insertion of promoters into the part to make a new regulated promoter. The promoter of most interest to us (not completed in this part) is a T7 promoter that is analogous to the pET vector systems. This part as it is present allows regulation of promoters (such as constitutive promoters) using CAP/lac elements, as well us the potential to upregulate a poorer promoter by CAP protein interactions with RNAPs. The part also allows promoters to more easily be visualized in 3A assembly on agarose gels.
Using the analogy of a T7 promoter with the target AflII and ClaI sites, the new promoter will have:
- The LacO/LacI binding site is a repressor site, inhibiting the RNA polymerase to transcribe the mRNA. By adding Lactose or its analog IPTG, the structure of the LacI repressor is changes, thus not being able to bind to the LacO site. this allows transcription to occur.
- Another regulatory element in the Lac system is the CAP binding site, which is located just upstream of the promoter region. Its role is to enhance transcription of the operon. The CAP protein is found throughout the cell in its inactive form - being activated by the molecule cAMP. When the levels of cAMP are high, there is more active CAP protein in the cell. Interestingly enough, the levels of cAMP are inversely proportional to the levels of glucose in the cell, so by depriving the cell from this carbon source the levels of transcription will thrive.
The T7 sequence is very small and though several designs of Lac/T7 systems have been designed in the parts registry, none have been made available in the distribution. When cloning T7 components, the small sequence means that there is large difficulty in gel electrophoresis detection and there is ambiguity as to whether a T7 promoter will work in the context of some promoter designs. Our part is based on the iGEM known part BBa_J04500 which is a functional Lac operon promoter (plus RBS) that has been benchmarked and tested in several iGEM projects, therefore providing a strong controllable and usable part for future iGEM distributions and another means to further characterise and control a promoter. A T7 insertion is potentially the most powerful promoter used in Synthetic Biology.
This part - as previously stated - was made from the part BBa_J04500. The first challenge that we encountered was to remove the current promoter elements found in the part: -35 and -10 elements. This was overcome using Quikchange XL Site-directed Mutagenesis to introduce unique restriction sites to allow the removal of the promoter region. The -35 element was mutated to include an AflII restriction site and the -10 element was mutated to a ClaI site. The T7 promoter was inserted via primers with ends to match the restriction sites, creating a new part when ligated into iGEM standard plasmid pSB1C3.
Part construction was confirmed through restriction digest and sequencing.
Source
The part BBa_J04500 was used as a template for the creation of this new part