Part:BBa_K2922039

The colicin-E1 operon under pBAD (Arabinose promoter) control with an amajLime chromoprotein reporte

Summary

This is a composite part consisting of an arabinose promoter (BBa_K206000), the CDS of Colicin-E1 (BBa_K2922024), the CDS of Colicin-E1 immunity protein (BBa_K2922025), the CDS of lysis protein (BBa_K2922026) and the amajLime chromoprotein reporter (BBa_K1033914). Each CDS has an RBS (BBa_B0034) behind. pBAD promoter could be induced by arabinose in Escherichia coli BL21 (DE3) strain and then express all proteins mentioned. E coli that can`t express Colicin-E1 immunity protein would be killed by Colicin-E1. Colicin-E1 immunity protein is used to protect itself from attack of extracellular Colicin-E1 and lysis protein helps the release of Colicin-E1. The amajLime chromoprotein reporter is used to present the growth curve specifically of strain which contains this part. This part is constructed in the aim of achieve our "spite" design.

The mechanism of E.coli BL21 (DE3) killing other bacteria by using Colicin-E1 kit.

Identification

In order to specifically record the growth curve of strains containing our colicin gene circuit, we combined BBa_K2922037 with amajLime chromoprotein reporter BBa_K1033914 to construct this part. This part was insert into pSB1C3 by standard assembly and transformed into E.coli BL21 (DE3) strain, Colonies were picked and cultured in liquid LB medium.

Fig.1 After 48h of culture, the medium(left) showed visible green, indicating that chromoprotein express successfully.

Growth curve of strains contained this part was detected by spectrophotometer. The values of OD600, OD577 and OD458 were detected and recorded, because OD600 are usually used to show the cell density of E.coli BL21 (DE3), 577 nm is the maximum emission wavelength of gfasPurple chromoprotein and 458 nm is the maximum emission wavelength of amajLime. The detail of the protocol can be viewed in Notebook-Experiment-Growth Curve：
https://2019.igem.org/Team:XMU-China/Experiments#

Fig.5 The comparison of the value of OD600 between experimental and control groups. PEF is the strain carring BBa_K2922039 and BBa_K1033917 is the strain carring BBa_K1033917 here. +TND/BBa_K1033914 is experimental group and TND/BBa_K1033914 is control group.

For more information, please go to our result:
https://2019.igem.org/Team:XMU-China/Results

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 125
  Illegal NheI site found at 2360
  Illegal NheI site found at 2383
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 65
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 642
  Illegal AgeI site found at 2000
  Illegal AgeI site found at 2057
  Illegal AgeI site found at 2189
  
• 1000
  COMPATIBLE WITH RFC[1000]