Part:BBa_K2922035
The colicin-N operon under pBAD(Arabinose promoter) control
Summary
This is a composite part consisting of an arabinose promoter (BBa_K206000), the CDS of Colicin-N (BBa_K2922027), the CDS of the immunity protein of Colicin-N (BBa_K2922028),and the CDS of lysis protein (BBa_K2922029). 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 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. This part is constructed in the aim of achieving our "Aggressive" design.
Ientification
During the time of buliding this circuit, we did the agarose gel electrophoresis experiment to verify. After the circuit was built, we sent the plasmid to sequence, and got the correct sequencing.
After new molecular cloning experiments, we did Enzyme-Cut identification to certify the plasmid is correct. We used the EcoRI and PstI to cut the plasmid, then we got two fragments - one is about 2200bp, and the other is about 5000bp(Fig.1). The fragment in 2200bp contained two fragments that one is linear pSB1C3 vector and the other is fragment of interest and they have similar length near 2200bp.
 Fig.1 The Agarose gel electrophoresis result for BBa_K2922035, plasmid that containing this part is digested by EcoRI and PstI.Strands in red box are fragments of interest. |
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In order to quantify its biological function, plasmid pSB1C3 was used to carry this part and this plasmid was transformed into E coli BL21 (DE3) strain. Colonies were picked and cultured in liquid LB medium, then we used the supernatant and precipitate in our inhibition zone experiment. The detail of the protocol can be viewed in Notebook-Experiment-Inhibition zone. (https://2019.igem.org/Team:XMU-China/Experiments#) Results of the inhibition zone are shown below.
 Fig.2 The inhibition zone test experiment of E coli BL21 (DE3) strain carrying BBa_K2922035. IPTG was added before for fully expression of colicin and immunity proteins. 100μL resuspended precipitate were added into oxford cup.Left area is experimental group and left area is control group. |
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This result indicated that colicin could be expressed and released by strain carrying BBa_K2922035, and it also showed the ability to kill other E coli strains without immunity proteins. The inhibition zone occurred in the control group means this promoter may be leaky.
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 125
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 65
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 696
Illegal AgeI site found at 1700 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
Each CDS are added an RBS (BBa_B0034) behind, and contain a terminator(BBa_B0010)
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