This part is coding for 2-haloacid dehalogenase, which can cleavage carbon-halogen bonds of halogenated compounds.
Through the 3A assembly, we successful assemble the HAD in the pSB1C3 vector. First, we amplify HAD through the PCR and through the gel electrophoresis to check the correct size.
Then clean up the PCR product and cut the HAD with EcoRI and PstI, and cut the pSB1C3 with EcoRI and PstI. Then another clean up for restriction enzyme product. Add ligase and buffer after calculate the nucleic acids concentration of the HAD and pSB1C3 and the ratio of the insert : vector. And the ligation is under 4℃ overnight. After the transformation, you can collect the colony overnight, and incubate in the LB to check if it is correct in the next day.
This is the result through the plasmid extraction, and the target place is at 2848bp, which is match to the result.
And also check again through the restriction enzyme digestion. The picture below is be cut by EcoRI and PstI. You can see there are two band, one is in 2070bp which refer to pSB1C3 and the other one is in 819bp, which is HAD.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21Illegal BglII site found at 684
- 23COMPATIBLE WITH RFC
- 25Illegal NgoMIV site found at 333
Illegal NgoMIV site found at 430
Illegal AgeI site found at 673
- 1000COMPATIBLE WITH RFC