Part:BBa_K1736300
LacI-LacIQ1
As explained in the project description & results page, the LacI-LacIQ1 was a late addition to the project to tackle the issue of toxicity to the expressing cells as a results of overexpression or over-synthesis of the enzymatic products. In this way, we add a layer of control to the expression of our ethene MO enzymes to ensure that they are expressing at appropriate and safe levels. Furthermore, its inactivation during the lab preparatory procedures of transformation and developing competent cells can aid the cell to deal with the immense amount of physiological stress imposed upon it and avoid an impending apoptosis. In order to ensure that we have a constitutively strong expression and activity of LacI, we placed the LacIQ1 promoter upstream of the E. coli LacI ORF, which is highly homologous and similar across bacteria. The following parts are already deposited for LacIQ1: BBa_K091112 and BBa_K091131. As for LacI, many parts for the gene have been deposited such as BBa_C0012. However, there is no composite part where the LacI gene and LacIQ1 have been placed together, making a new addition to the registry which will be useful for future and teams and experiments relying on strong expression control of gene under lac operon regulation. To confirm that the composite part is functional, we ran an experiment to qualitative check for the activity of LacI via the reporter gene LacZ and it's hydrolysis of X-gal (derivative of lactose which turns blue upon hydrolysis by B-galatosidase encoded by LacZ-alpha, and is not taken up by the bacteria). E. coli competent cells containing the LacZ-alpha gene (placed under the control of lac promoter) were transformed with LacI-LacIQ1 and placed in media containing IPTG and no IPTG to induce expression of LacZ (B-galactosidase), with both having equal amounts of X-gal.
After the induction, there were blue colonies on the IPTG media as expected, however, some blue colonies (to a lesser extent qualitatively) in the media containing no IPTG. This is unexpected and hypothesised to be due to the genomic native host lac operon system being responsible for the B-galactisidase activity and expression. This explanation becomes more credible considering that the host contains a lac operon containing both fragments of B-galactosidase enzyme LacZ-alpha and LacZ-beta, where each are under the control of a separate lac promoter. Hence, due to the high-copy presence of LacZ-alpha in a recombiannt vector in the cells and the higher expression of repressor proteins (also due to the high copy number of recombinant vectors containing LacI-LacIQ1), most of the repressor protein block the LacZ-alpha on the vectors, not the genomic ones. Furthermore, it could also be possible that the LacI repressor cannot control the genomic LacZ-beta expression, which can still be expressed and perform X-gal hydrolysis. Hence, while we know this system should work due to countless studies including a paper on LacIQ1 by Glascock CB et al. 3, every user of this construct has to check for the absence of LacZ subunits in the genome which could breakaway from the claws of LacI repressor control.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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