Coding

Part:BBa_K2546002

Designed by: Tsu-Wang Sun   Group: iGEM18_NCHU_Taichung   (2018-10-03)


2-haloacid dehalogenase plus laccase with robosome binding site

This part is the combination of BBa_K2546001 and BBa_K2546011 which contain ribosome binding site.

RESULT

Through the 3A assembly, we successful assemble the HL(HAD+LACCASE). There is a ribosome binding site in front of the both gene. And the ribosome is come from BBa_B0034. (The 3A assembly protocol you can see in here.) Through the PCR to amplify our target gene, then clean up the PCR product. Cut with EcoRI and PstI in the same micro-eppendorf ,and add T4 ligase and buffer after the restriction enzyme digestion. The reaction environment is 4℃ overnight.

caption

After incubate the transformed colony in LB ,we extract the plasmid. Our target gene is 4525bp. As for the picture above, you can see a band located above the 4000bp. Also, through the PCR amplification, you can see the left band is whole gene of HAD+LACCASE and is located between 2000bp and 3000bp. The middle band is only HAD and is also match to the ideal size. The last one is Laccase, which is 1590bp.

caption


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 702
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 351
    Illegal NgoMIV site found at 448
    Illegal NgoMIV site found at 1173
    Illegal AgeI site found at 691
    Illegal AgeI site found at 1683
    Illegal AgeI site found at 2067
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
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