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
Other teams (Slovenia 2010, Cambridge 2009) had characterized arabinos induced promoters.
Given the complexity of our system, we needed to guarantee an effective expression of our constructs with an optimal concentration for induction by L-arabinose.
The protocol was taken from Team Cambridge 2009 and Tec-Monterrey 2010.
Pick three different colonies that contain the desired part to be characterized and place each of them in a different 50 mL tube with 5 mL LB medium.
Add 1 μL of the corresponding antibiotic per each mL of LB medium.
Pick one colony that does not contain the plasmid with the part that will be characterized (control) and place it in a 50 mL tube with 5 mL LB medium.
Incubate all the 4 tubes for 16 hours on a shaker at 37°C and 350 rpm.
Check the OD600 of the cultures and dilute with fresh LB medium until an OD600 of 0.1 is reached. Make sure that you have at least 7 mL of each culture.
Add 1 μL of the corresponding antibiotic per each mL of LB medium.
Incubate all the 4 tubes on a shaker at 37°C and 350 rpm until an OD600 of 0.6 is reached.
Fill each well with 198 μl of inoculum and 2 μl of L-Arabinose at different concentrations. Make 3 repetitions of each colony with the different concentrations of L-Arabinose.
The microplate is then read by the microplate reader by using the following protocol: Set temperature to 37°C
Kinetic reading lasting 4 hours with measurements every 5 minutes.
Absorbance (600 nm filter) and Fluorescence (Excitation: 485 nm, Emission: 528 nm).
Shaking in intensity 2 for 5 seconds before every reading.
Export the results of the well data to an Excel sheet for further interpretation.
The vehicle of expression was BW2773 strain, recommended for its inability to metabolize arabinose.
GRAFICAAAAAA:O!!!!!
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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