Part:BBa_K633015
AraC+Pbad promoter+RBS+ Ompa&lpp signal peptide+Sacc
This is a composite device consisting of the following parts: araC (BBa_I13458), pBad Strong (BBa_K20600), a ribosome binding site (BBa_B0034), OmpA outer protein membrane A fused to linker (BBa_K103006), together with a new part we submitted: SacC extracellular succrase (BBa_K633003). The function of this construct is to send the chimeric protein OmpA + Succrase to the periplasm when expression is induced with L-arabinose.
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).
Figure 1
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.
SacC is active in whole cell assays!
Figure 2. SacC Activity Assay Graph
Whole cells of E. coli strain (BL21SI) +sacC+ompA produced a fructose concentration of 350.71±60.97 µM which is 149.36 µM higher than the negative control cells (Figure 2), 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 indicator that the enzyme is active in the outer membrane of E. coli.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1247
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1187
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 979
Illegal AgeI site found at 2657 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 2357
Illegal SapI site found at 961
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