Part:BBa_K2262015

Constitutive Promoter + RBS + fMt

Figure 1. J23119+RBS+fMt

Introduction

      This sequence is designed for constitutively chelating arsenic ions. We ligated a constitutive promoter(BBa_J23119) with metallothionein (fMt, BBa_K190019) to produce arsenic-binding protein. This metallothionein (fMt) is a kind of chelating protein from Fucus vesiculosus. It can bind both Arsenite (III) and Arsenate(V). This part was first designed by Groningen of iGEM 2009. The part of K190019 consists of fMt and RBS(ribosome--‐binding Site, for initiating translation).

Modifying and Improving the Existed Biobrick

Previous biobrick BBa_K190031 of 2009 iGEM10_Groningen

      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 2.) Thus, we decided to modify the biobrick by ligating fMt(BBa_K190019) with another constitutive promoter (BBa_J23119). The Figure 3 shows the electrophoresis of BBa_K190019 when its plasmid was cut by Xba I and Pst I. And the Figure 4 shows the electrophoresis of BBa_J23119 and BBa_J23109 when their plasmids were cut by Spe I and Pst I.

Figure 2. The sequence of J23109 Plasmid.

Figure 3. The electrophoresis of BBa_K190019.

Figure 4. The electrophoresis of BBa_J23119 and BBa_J23109.

Result

1.

      We first examined the growth curve of E. coli DH5Alpha in arsenic solution. We compared the growth curve of E. coli DH5Alpha in arsenic solution with that curve in solution without arsenic ions. The Table 1 shows the experimental design for the growth curve of E. coli DH5Alpha.

Table 1.The experiment design for the growth curve of E. coli DH5Alpha.

      The results showed that the growth of E.coli DH5Alpha won’t be affected by the arsenic concentration below 100ppm.(Table 2)

Table 2.The growth of E.coli DH5Alpha in different conditions.

2.

      We examined the growth curve E. coli DH5Alpha equipped with fMt plasmid in different concentration of arsenate. The Table 3 shows the experimental design for the growth curve of E. coli DH5Alpha with fMt plasmid.

Table 3.The experiment design for the growth curve of E. coli DH5Alpha with fMt plasmid.

      The results of this experiment indicate that E. coli DH5Alpha containing the transformed plasmid can survive in arsenic concentrations from 1 ppm to 100 ppm(Table 4)

Table 4.The growth of E.coli DH5Alpha 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 does not affect the arsenic concentration.

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]