Part:BBa_K1104100:Experience

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Experiment

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

mngB clonning result
　　Before doing the functional test, it is a must that we have to construct a circuit that can constitutively produce alpha-mannosidase. We have added a constitutive promoter, BBa_J23102,at the upper stream of the mngB coding sequenceand terminator behind the gene. The whole length is 3,146bp. In the right well of the picture at the right is the marker and the left is our part, which shows that the insert of mngB with promoter, RBS and terminator is correct, showing that we have successfully finish our circuit.

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