Difference between revisions of "Part:BBa K2922035"
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===Ientification=== | ===Ientification=== | ||
− | + | When we are building this circuit, we are doing the agarose gel electrophoresis experiment to verify. After the circuit is built, we send the plasmid to sequence, and get the correct sequencing. | |
− | + | <br> | |
+ | After new molecular cloning experiments, we do Enzyme-Cut identification to certify the plasmid is correct. We use the <i>EcoR</i>I and <i>Pst</i>I to cut the plasmid, then we getting two fragments - one is about 2200bp, and the other is about 5000bp(Fig.1). The fragment in 2200bp may contained two fragments that one is linear pSB1C3 vector and the other is fragment of interest and they have similar length near 2200bp. | ||
+ | |||
<table><tr><th>[[Image:PNAGE.png|thumb|Fig.1 The Agarose gel electrophoresis result for <partinfo>BBa_K2922035</partinfo>, plasmid that containing this part is digested by <i>EcoR</i>I and <i>Pst</i>I.Strands in red box are fragments of interest.]]</th><th></table> | <table><tr><th>[[Image:PNAGE.png|thumb|Fig.1 The Agarose gel electrophoresis result for <partinfo>BBa_K2922035</partinfo>, plasmid that containing this part is digested by <i>EcoR</i>I and <i>Pst</i>I.Strands in red box are fragments of interest.]]</th><th></table> | ||
Revision as of 10:33, 19 October 2019
The colicin-N operon under pBAD(Arabinose promoter) control
Summary
This is a composite part consist of an arabinose promoter (BBa_K206000), CDS of colicin-N (BBa_K2922027), CDS of colicin-N immunity protein (BBa_K2922028) and CDS of lysis protein (BBa_K2922029). Each CDS has an RBS (BBa_B0034) behind. Arabinose 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-N immunity protein would be killed by colicin-N, colicin-N immunity protein is used to protect itself from attack of extracellular colicin-N and lysis protein helps the release of colicin-N. This part is constructed in the aim of achieve our “aggressive” design.
Ientification
When we are building this circuit, we are doing the agarose gel electrophoresis experiment to verify. After the circuit is built, we send the plasmid to sequence, and get the correct sequencing.
After new molecular cloning experiments, we do Enzyme-Cut identification to certify the plasmid is correct. We use the EcoRI and PstI to cut the plasmid, then we getting two fragments - one is about 2200bp, and the other is about 5000bp(Fig.1). The fragment in 2200bp may contained two fragments that one is linear pSB1C3 vector and the other is fragment of interest and they have similar length near 2200bp.
In order to quantify its biological function, Plasmid pSB1C3 are used to carried this part and this plasmid are transformed into E coli BL21 (DE3) strain. Clonies 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. Results of the inhibition zone are shown below.
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
- 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)