Part:BBa_K633014
AraC+Pbad promoter+RBS+signal peptide phoA+Cellulase+Linker+estA membrane protein
The composite part includes a functional expression device for autotransporter membrane protein estA, with a linker united to a cellulase, attached to a membrane signaling peptide. This was our final construct missing a terminator. By the use of the restriction enzymes, NheI and SacI, more enzymes can be displayed outside the outer membrane of E. coli.
The construct efectiveness was tested by an enzymatic assay, The results can be seen here: http://2011.igem.org/Team:Tec-Monterrey/projectresults
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-Cell CelD+estA Activity
Figure 2. Whole-Cell Cellulase Activity.
The glucose concentration in celD + estA strain was of 332.04 µM and in the Negative Control (C-) was of 275.85 µM that is a difference in the glucose concentration of 57 µM. The result of the t- test was the rejection of the null hypothesis, suggesting that the difference between them is significant.
Cell-Lysate CelD+estA Activity
Figure 3.Cellulase Activity of Cell lysates.
In the cell-lysate cellulase activity assay (Figure 3) The glucose concentration in the soluble fraction of celD-estA was of 358 µM and in the Negative Control (C-) was of 323 µM.In the insoluble fraction, the glucose contentration of the celD-estA was 374 µM and in the Negative Control (C-) was of 264 µM. The difference in soluble and insoluble fractions with its negative control was 35 µM while the difference in the insoluble fraction was 110 µM. The result of the t-test was the rejection of the null hyphothesis, suggesting that the difference between them is also significant.
Note: There is an ilegal NheI restrition site in the promoter region, part BBa_K206000.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1247
Illegal NheI site found at 3168 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 1187
Illegal BamHI site found at 1981 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1845
Illegal NgoMIV site found at 3398
Illegal AgeI site found at 979
Illegal AgeI site found at 1488
Illegal AgeI site found at 1904
Illegal AgeI site found at 2733 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 961
//cds/membrane
//function/degradation/cellulose
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