Part:BBa_K633014

AraC+Pbad promoter+RBS+signal peptide phoA+Cellulase+Linker+estA membrane protein

The composite part includes a functional expression vector 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.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1247
Illegal NheI site found at 3168
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 1187
Illegal BamHI site found at 1981
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE 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
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI site found at 961