Coding

Part:BBa_K190031

Designed by: Wilfred Poppinga   Group: iGEM09_Groningen   (2009-09-02)

fMT with low constitutive promoter

The metallothionein fMT under control of a low constitutive promotor BBa_J23109


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
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 81
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 170
  • 1000
    COMPATIBLE WITH RFC[1000]



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Contribution

Group: NCTU_Formosa 2017

Author: Yi-Ting Lin, Huei-Yu Yeh, Sheng-Ping Chu

New improved part BBa_K2262015


Summary
The metallothionein (BBa_K190031) is a fMt(BBa_K190019) under control of a low constitutive promotor (BBa_J23109). We failed several times in replicating the ligation of these two parts. After sequencing BBa_K190031, BBa_K190019, and BBa_J23109, we found the constitutive promoter BBa_J23109 has two Spel restriction sites in the prefix.(Figure 1.) Thus, we decided to modify the biobrick by ligating fMt(BBa_K190019) with another constitutive promoter (BBa_J23119).

Figure 1. The sequence of J23109 Plasmid.

Result


1.


      We first examined the growth curve of E. coli DH5α in arsenic solution. We compared the growth curve of E. coli DH5α in arsenic solution with that curve in solution without arsenic ions. Table 1 shows the experimental design for the growth curve of E. coli DH5α.


Table 1.The experiment design for the growth curve of E. coli DH5α.




      The results showed that the growth of E. coli DH5α won’t be affected by the arsenic concentration below 100ppm.(Table 2)

Table 2.The growth of E. coli DH5α in different conditions.




2.

      We examined the growth curve f E. coli DH5α equipped with fMt plasmid in different concentration of arsenate. Table 3 shows the experimental design for the growth curve of E. coli DH5α with fMt plasmid.


Table 3.The experiment design for the growth curve of E. coli DH5α with fMt plasmid.




      The results of this experiment indicate that E. coli DH5α containing the transformed plasmid can survive in arsenic concentrations from 1 ppm to 100 ppm(Table 4).

Table 4.The growth of E. coli DH5α in different conditions.




      In conclusion, we modified the part of BBa_K190031 by replacing the promoter BBa_J23109 by BBa_J23119. The growth of E. coli with this new plasmid is not affected the arsenic concentration.


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