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.
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.
The Escherichia coli strain, Rosetta Gami, was choosen as a host for the chimeric protein because it has an improved folding system.
We used the IUPAC Filter Paper Assay, to determine the activity of the cellulase. This method is based in the reduction of the DNS, generating a proportional colorimetric concentration.
The assay was assest to the whole-cells, but also, we lysated the cells and separated them in two main fractions: soluble and insoluble. We were expecting more activity in the insoluble because our protein has a transmembranal domain.
The negative controls of the assay were non-transformed cells.
All samples were treated equally in the assay.
In the “ Whole-cell cellulase Activity” chart, is observed that there is a difference in the glucose concentration between the Negative Control cells (C-) and the CelD+ estA cells. A t-student was done (sigma = 0.05) . The result was the rejection of the null hypothesis (Ho), this meant that the difference was significant.
In the “Cellulase Activity Cell Lysates” is analysed two fractions : Soluble ans Insoluble. As it can be seen, in both cases, there is a difference between the celD+estA cells and their respective Negative Controls (C-) . Also, it is noticed that there is a higher difference in the Insoluble fraction that in the Soluble one. A t-student was done (sigma = 0.05). The result was the rejection of the null hypothesis (Ho), this meant that the difference was significant.
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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