Difference between revisions of "Part:BBa K1104100:Design"

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==mannosidase function test==
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==Mannosidase function test==
 
  After cloning the mngB gene into the ''E. coli'', we would want to know whether our part work. So we decided to extract alpha-mannosidase from the cytoplasm of the ''E. coli'' and test its function by directly interact with 2-O-(6-phospho-α-D-mannosyl)-D-glycerate, and through HPLC to see if it can successfully turn it into α-D-mannose-6-phosphate and D-glycerate.Experimental process is listed below:
 
  After cloning the mngB gene into the ''E. coli'', we would want to know whether our part work. So we decided to extract alpha-mannosidase from the cytoplasm of the ''E. coli'' and test its function by directly interact with 2-O-(6-phospho-α-D-mannosyl)-D-glycerate, and through HPLC to see if it can successfully turn it into α-D-mannose-6-phosphate and D-glycerate.Experimental process is listed below:
 
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Revision as of 17:26, 27 September 2013

mngB - alpha-mannosidase


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 716
    Illegal BglII site found at 992
    Illegal BglII site found at 2139
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 235
    Illegal AgeI site found at 2242
  • 1000
    COMPATIBLE WITH RFC[1000]


Design

alpha mannosidase primer:
  Since the mngB comes direct from E. coli substrain MG1655, we have designed a primer to clone from the whole genome of MG1655.The primer we used is listed below:

primer sequence whole primer temp. binding part temp. GC%
forward: ctg GAATTCGCGGCCGCTTCTAG atgAAAGCAGTATCTCGCGTTCACATCACCCCG 76℃ 68℃ 55%
reverse: gga CTGCAGCGGCCGCTACTAGTA tcaGGCAAGCCGGTAACTGAACGTCCG 76℃ 68℃ 58%

Mannosidase function test

  After cloning the mngB gene into the E. coli, we would want to know whether our part work. So we decided to extract alpha-mannosidase from the cytoplasm of the E. coli and test its function by directly interact with 2-O-(6-phospho-α-D-mannosyl)-D-glycerate, and through HPLC to see if it can successfully turn it into α-D-mannose-6-phosphate and D-glycerate.Experimental process is listed below:
1.use Continuous High Pressure Cell Disrupter to crush the E. coli that produce alpha-mannosidase
2.filter the product and save the filtrate in a eppendorf
3.add 2-O-(6-phospho-α-D-mannosyl)-D-glycerate into eppendorf
4.incubate at 25℃ for 30 min.
5.run HPLC and compare it with the two standard line of only 2-O-(6-phospho-α-D-mannosyl)-D-glycerate and with only the extraction liquid of E. coli cytoplasm
6.compare the area of the data lines with the area of the standard line to calculate the efficiency of the alpha-mannosidase produced by E. coli


Source

E. coli, K12, MG1655

References