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

(→‎Design)
(→‎Mannosidase function test)
 
(4 intermediate revisions by the same user not shown)
Line 27: Line 27:
  
 
==Mannosidase function test==
 
==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 mngB gene into ''E. coli'', it is a must to know whether our part is well-constructed and well perform. Therefore, we decide to extract alpha-mannosidase from the cytoplasm of ''E. coli'', and test its function by directly interact with 2-O-(6-phospho-α-D-mannosyl)-D-glycerate.  With the aid of HPLC, we can evidently realize whether our part is able to turn it into α-D-mannose-6-phosphate and D-glycerate.
−
<br>
+
　　
−
1.use Continuous High Pressure Cell Disrupter to crush the ''E. coli'' that produce alpha-mannosidase <br>
+
 
−
2.filter the product and save the filtrate in a eppendorf<br>
+
　　Experimental process is listed below:
−
3.add 2-O-(6-phospho-α-D-mannosyl)-D-glycerate into eppendorf <br>
+
 
−
4.incubate at 25℃ for 30 min.<br>
+
1.Use Continuous High Pressure Cell Disrupter to crush the ''E. coli'' that produce alpha-mannosidase <br>
−
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 <br>
+
2.Filter the product and save the filtrate in a eppendorf<br>
−
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''
+
3.Add 2-O-(6-phospho-α-D-mannosyl)-D-glycerate into an eppendorf <br>
 +
4.Incubate at 25℃ for 30 min.<br>
 +
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 <br>
 +
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''
  
  

Latest revision as of 17:38, 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 directly from E. coli substrain MG1655, we have designed a primer to clone from the whole genome of MG1655. The primer we use 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 mngB gene into E. coli, it is a must to know whether our part is well-constructed and well perform. Therefore, we decide to extract alpha-mannosidase from the cytoplasm of E. coli, and test its function by directly interact with 2-O-(6-phospho-α-D-mannosyl)-D-glycerate. With the aid of HPLC, we can evidently realize whether our part is able to 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 an 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