Difference between revisions of "Part:BBa K633015"
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<partinfo>BBa_K633015 short</partinfo>
 
<partinfo>BBa_K633015 short</partinfo>
  
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Other teams (Slovenia 2010, Cambridge 2009) had characterized arabinos induced promoters.
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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).  
  
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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.
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[[Image:Induction_gfp.jpg]]
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'''The protocol was taken from Team Cambridge 2009 and Tec-Monterrey 2010.'''
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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.
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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.
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Add 1 μL of the corresponding antibiotic per each mL of LB medium.
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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.
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Incubate all the 4 tubes for 16 hours on a shaker at 37°C and 350 rpm.
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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.
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Add 1 μL of the corresponding antibiotic per each mL of LB medium.
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Incubate all the 4 tubes on a shaker at 37°C and 350 rpm until an OD600 of 0.6 is reached.
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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.
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The microplate is then read by the microplate reader by using the following protocol:
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Set temperature to 37°C
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Kinetic reading lasting 4 hours with measurements every 5 minutes.
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Absorbance (600 nm filter) and Fluorescence (Excitation: 485 nm, Emission: 528 nm).
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Shaking in intensity 2 for 5 seconds before every reading.
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Export the results of the well data to an Excel sheet for further interpretation.
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The vehicle of expression was BW2773 strain, recommended for its inability to metabolize arabinose.
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GRAFICAAAAAA:O!!!!!
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 14:54, 17 October 2011

AraC+Pbad promoter+RBS+ Ompa&lpp signal peptide+Sacc

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).

Induction gfp.jpg

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
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1187
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
    Illegal AgeI site found at 2657
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2357
    Illegal SapI site found at 961