Composite

Part:BBa_K2922039

Designed by: Gezhi Xiao   Group: iGEM19_XMU-China   (2019-10-13)


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.

To examine the virulence of colicin kit, Co-culture experiment between strain carrying BBa_K2922039 and strain carrying gfasPurple chromoprotein is carried out. The values of OD600, OD577 and OD458 were detected by spectrophotometer 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#
Results are shown below:

Fig.2 The co-cultural growth curve of E.coli BL21 (DE3) strains which contained BBa_K2922039 or BBa_K1033917. The values of OD600, OD577 and OD458 were detected to specifically track each strain`s growth status. Arabinose is added in 2h to induced the expression of colicin.
Fig.3 The co-cultural growth curve of E.coli BL21 (DE3) strains which contained BBa_K2922039 and BBa_K1033917. The values of OD600, OD577 and OD458 were detected to specifically track each strain’s growth status. These is the result of control group which arabinose was not added.
Fig.4 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. +PEF/BBa_K1033917 is experimental group and -PEF/BBa_K1033917 is control group.

These results indicate that interference from E.coli cell density to measured values of OD577 and OD458 is beyond our expectation. Therefore, before removing the interference from E.coli cell density, there are some problems in using chromoproteins to specifically track the growth curve of a strain in co-culture system. The comparison of OD600 between experimental group and control group do not shown significance difference, which means the Coliicin-E1 may not expressed or it is inactive by unknown reason.In conclusion, a further study of relationship between cell density and chromoprotein concentration is needed for improving our experiment, and we need further examination for our Colicin-E1 kit in co-culture experiments to make sure it could work properly.


To futher investigate the relationship between E.coli cell density and concentration of chromoprotein, it is quite necessary to verify the influence from cell density to measured value of absorbance in 577nm or 458nm. Because we need this data to quantify the concentration of chromoprotein in order to specifically record the growth status of a strain carrying BBa_K2922039 or BBa_K1033917. Standard curve of E.coli BL21 strain carrying BBa_K2922039 is constructed by dilution. Results is shown below:

Fig.5 The standard curve for strain contained BBa_K2922039, which is used to shown the relationship between cell density(indicate from OD600) and measured value of OD577 and OD458, in order to know the relationship between cell density and concentration of chromoprotein.

The growth status for a given strain expressing chromoprotein in a co-culture system could be derive from original growth curve by data processing. Result are shown in our demonstrate page (https://2019.igem.org/Team:XMU-China/Demonstrate), and protocol is included in appendix.


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]


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