Part:BBa_K2922038
The colicin-N operon under T7 promoter control with a gfasPurple chromoprotein reporter
Summary
This is a composite part consisting of a T7 promoter (BBa_K525998), the CDS of Colicin-N (BBa_K2922027), the CDS the immunity protein of Colicin-N (BBa_K2922028), the CDS of lysis protein (BBa_K2922029) and the gfasPurple chromoprotein reporter (BBa_K1033917). Each CDS has an RBS (BBa_B0034) behind. T7 promoter could be induced by IPTG or lactose in Escherichia coli BL21 (DE3) strain and then express all proteins mentioned. E.coli that can not express the immunity protein of Colicin-N would be killed by Colicin-N. Colicin-N immunity protein is used to protect itself from the attack of extracellular Colicin-N and lysis protein helps the release of Colicin-N. The gfasPurple chromoprotein reporter is used to present the growth curve specifically of the strain which contains this part. This part is constructed in the aim of achieving our "Aggressive" design.
Ientification
In order to specifically record the growth curve of strains containing our colicin gene circuit, we combined BBa_K2922034 with gfasPurple chromoprotein reporter BBa_K1033917 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.
This result indicates that concentration of chromoprotein gfasPurple (shown by OD577) has certain relationship with cell density of E.coli BL21 (DE3) (shown by OD600), which means it could be expressed and degraded by E.coli BL21 (DE3) strain and it has the potential to specifically record the growth status of strains expressing the protein. Otherwise, the value of OD458 showed a similar trend with the value of OD577, although there was no amajLime chromoprotein exists, which means E.coli BL21 (DE3) cell has interference in measuring the value of OD577 and OD458. In order to specifically record the growth status of a strain contained chromoprotein, we need further investigation of the relationship between cell density and chromoprotein concentration.
To examine the virulence of colicin kit, Co-culture experiment between strain carrying BBa_K2922038 and strain carrying amajLime 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_K2922038 or BBa_K1033914. The values of OD600, OD577 and OD458 were detected to specifically track each strain`s growth status. IPTG 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_K2922038 and BBa_K1033914. 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 IPTG was not added. |
|---|
 Fig.4 The comparison of the value of OD600 between experimental and control groups. TND is the strain carring BBa_K2922038 and BBa_K1033914 is the strain carring BBa_K1033914 here. +TND/BBa_K1033914 is experimental group and -TND/BBa_K1033914 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. But the cell density in experimental group was significantly lower than control group, which means the colicin kit was active successfully by IPTG induced and indicator’s growth was inhibited. In conclusion, the growth curve primary showed the virulence of colicin protein but a further study of relationship between cell density and chromoprotein concentration is needed for improving our experiment.
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_K2922038 or BBa_K1033914. Standard curve of E.coli BL21 strain carrying BBa_K2922038 is constructed by dilution. Results is shown below:
 Fig.5 The standard curve for strain contained BBa_K2922038, 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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1938
Illegal NheI site found at 1961 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 598
Illegal AgeI site found at 1602 - 1000COMPATIBLE WITH RFC[1000]
| None |