Difference between revisions of "Part:BBa K633015"

Revision as of 16:06, 17 October 2011

AraC+Pbad promoter+RBS+ Ompa&lpp signal peptide+Sacc

In order to find suitable arabinose concentrations to induce expression of the construct, the protein coding sequences were substituted by a GFP reporter, BBa_e0040. A double terminator, BBa_e0014, was added as well. The expression vehicle was Escherichia coli, BW27783 strain, due to its inability to metabolize arabinose. Fluorescence was measured in transformed E. coli cultures induced with different concentrations of L-arabinose (1%), following the protocol used by Cambridge (2009) and Tec-Monterrey (2010).

The chart shows that at low concentrations of arabinose, poor induction levels are obtained since fluorescence changes very little over time. A noticeable increase of fluorescence is viewed from a concentration of 100 µM onwards. Similar results were found in BBa_I0500 characterization by Cambridge (2011) and Groningen (2011), proving further the effectiveness of using this concentration of arabinose as a minimum to induce expression of the construct.

Whole cells of E. coli strain (BL21SI) +sacC+ompA produced a fructose concentration of 350.71±60.97 uM which is 149.36 uM higher than the negative control cells (Figure), a T-test with 2 tails and alpha value of 0.05 was carried out, and the null hypothesys of "the population means are the same" was rejected, indicating that there is difference between the fructose concentration in the control strain and those of the sample strain. And although further investigation is required, the evidence we have is a strong evidence that the enzyme is active in the outer membrane of E. coli. Further research should be focused on SDS-PAGE with more efficient staining/blotting technique, expression of sacC fusing it with estA protein fragments, and more experiments of sacC enzymatic assay.

Sequence and Features