Coding

Part:BBa_K2546006

Designed by: Tsu-Wang Sun   Group: iGEM18_NCHU_Taichung   (2018-10-09)


TonB + 2-haloacid dehalogenase + laccase

This part is the combination of BBa_K2546012 and BBa_K2546002, which is our ultimate goal to let three gene be expressed in one circuit. After successfully assemble this circuit, we also do the growth curve test with TCDD and without TCDD.


RESULT

After amplify BBa_K2546012 and clean up the PCR product, we cut BBa_K2546012 with EcoRI and SpeI, and also cut BBa_K2546002 with EcoRI and XbaI .After cut BBa_K2546012 with EcoRI and SpeI, need to do gel extraction (you can see the protocol in 3A assembly and Gibson). Calculating the ratio of insert : vector is important in ligation. Till the colony is growth on the CM plate, then incubate in LB 37℃ overnight. In the next day, we extract the plasmid. Our ideal plasmid size is 6795bp, and the band is located in nearly above 6000bp, so we do the restriction enzyme cut. Here is the result.

caption
caption

The first band is cut with EcoRI and the second band is cut with the XbaI, these two bands are all cut with one restriction side, and the band is identical and is located above the 6000bp ,which is similar to 6795bp.

caption

We also cut with ApaI, which has two restriction side in the plasmid and is 3979bp and 2822bp respectively. Observing from the picture above, you can see there is two band, and is similar to 3979bp and 2822bp. In conclusion, we successful to let three gene being express in the same circuit.




the Growth Curve Test


TonB+HAD+TLcc (J23100) had better growth in the medium with TCDD when TCDD concentration is under 0.2ppm (Fig. 1C) contrast to the growing of the control J23100 (Fig. 1A).

Legends
T--NCHU Taichung--part13.jpeg

Fig.1A

T--NCHU Taichung--part15.jpeg

Fig.1C

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
    Illegal NheI site found at 2252
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 3015
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2664
    Illegal NgoMIV site found at 2761
    Illegal NgoMIV site found at 3486
    Illegal AgeI site found at 3004
    Illegal AgeI site found at 3996
    Illegal AgeI site found at 4380
  • 1000
    COMPATIBLE WITH RFC[1000]


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