Difference between revisions of "Part:BBa K2922035"
Line 3: Line 3:
  
 
===Summary===
 
===Summary===
−
This is a composite part consist of an arabinose promoter (<partinfo>BBa_K206000</partinfo>), CDS of colicin-N (<partinfo>BBa_K2922027</partinfo>), CDS of colicin-N immunity protein (<partinfo>BBa_K2922028</partinfo>) and CDS of lysis protein (<partinfo>BBa_K2922029</partinfo>). Each CDS has an RBS (<partinfo>BBa_B0034</partinfo>) behind. Arabinose promoter could be induced by Arabinose in <i>Escherichia coli</i> BL21 (DE3) strain and then express all proteins mentioned. <i>E coli</i> 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 “spite” design.
+
This is a composite part consisting of an arabinose promoter (<partinfo>BBa_K206000</partinfo>), the CDS of Colicin-E1 (<partinfo>BBa_K2922027</partinfo>), the CDS of the immunity protein of Colicin-E1 (<partinfo>BBa_K2922028</partinfo>),and the CDS of lysis protein (<partinfo>BBa_K2922029</partinfo>). Each CDS has an RBS (<partinfo>BBa_B0034</partinfo>) behind. pBAD promoter could be induced by arabinose in <i>Escherichia coli</i> BL21 (DE3) strain and then express all proteins mentioned. <i>E.coli</i> that can`t express the immunity protein of Colicin-E1 would be killed by Colicin-E1, Colicin-E1 immunity protein is used to protect itself from the attack of extracellular Colicin-E1 and lysis protein helps the release of Colicin-E1. This part is constructed in the aim of achieving our "Aggressive" design.
  
 
<table><tr><th>[[Image:Ndesign.png|thumb|800px|The diagram of Colicin-N kit design]]</th><th></table>
 
<table><tr><th>[[Image:Ndesign.png|thumb|800px|The diagram of Colicin-N kit design]]</th><th></table>

Revision as of 13:43, 19 October 2019

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-E1 (BBa_K2922027), the CDS of the immunity protein of Colicin-E1 (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-E1 would be killed by Colicin-E1, Colicin-E1 immunity protein is used to protect itself from the attack of extracellular Colicin-E1 and lysis protein helps the release of Colicin-E1. This part is constructed in the aim of achieving our "Aggressive" design.

The diagram of Colicin-N kit 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.

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.

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.

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.

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


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 125
  • 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 696
    Illegal AgeI site found at 1700
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Each CDS are added an RBS (BBa_B0034) behind, and contain a terminator(BBa_B0010)