Difference between revisions of "Team:JNFLS/Parts"

 
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<h3>Our Parts </h3>
  
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<p>Our project is to develop a biosensor based on the aptamer target on the HCV core protein, using competition based rolling amplification. This device could be used for the detection of trace HCV virus in unpaid donation blood test and blood transfusion in the clinic, which could shorten the window period of HCV detection. In order to achieve enough HCV C protein expressed in Prokaryotes, we constructed some recombinant plasmids of HCV C gene, some of them are truncated or/and optimized, some of them are tagged with TEE and 6×His marker. We cloned all these modified HCV C genes into pSB1C3 plasmid backbone to produce new BioBrick parts, which are compatible with RFC 10. All those parts worked well, particularly BBa_K2608004, which was selected to use in our project. </p>
  
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<h3>Part Table </h3>
 
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<p>Our project is to develop a biosensor based on the aptamer target on the HCV core protein, using competition based rolling amplification. This device could be used for the detection of trace HCV virus in unpaid donation blood test and blood transfusion in the clinic, which could shorten the window period of HCV detection. In order to achieve enough HCV C protein expressed in Prokaryotes, we constructed some recombinant plasmids of HCV C gene, some of them are truncated or/and optimized, some of them are tagged with TEE and 6×His marker. We cloned all these modified HCV C genes into pSB1C3 plasmid backbone to produce new BioBrick parts, which are compatible with RFC 10. All those parts worked well, particularly BBa_K2608004, which was selected to use in our project. </p>
 
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<p>HCV is a single strand RNA virus with a coat. Core antigen is the main protein of nucleocapsid. It is an important part of the virus particle. Core protein can control the virus itself, as well as the interactions of virus and host cells. After signal peptide was inserted to the 5’-UTR of HCV C gene, the expressed fusion protein can be secreted to the outside of the cell, with the ability of self-assembly into the virus particle. Ii is a very good tool for HCV research. All the part genes were inserted into the vector plasmid pColdⅡ, showed as below.</p>
 
<p>HCV is a single strand RNA virus with a coat. Core antigen is the main protein of nucleocapsid. It is an important part of the virus particle. Core protein can control the virus itself, as well as the interactions of virus and host cells. After signal peptide was inserted to the 5’-UTR of HCV C gene, the expressed fusion protein can be secreted to the outside of the cell, with the ability of self-assembly into the virus particle. Ii is a very good tool for HCV research. All the part genes were inserted into the vector plasmid pColdⅡ, showed as below.</p>
 
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<b>Fig.1 Map of pCold Ⅱvector shows TEE and His tag are inserted to the upstream of multiple cloning sites.</b>
 
<b>Fig.1 Map of pCold Ⅱvector shows TEE and His tag are inserted to the upstream of multiple cloning sites.</b>
 
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<b>Fig.2 Diagram of pCold Ⅱvector shows the cloning site of HCV C gene insertion.</b>
 
<b>Fig.2 Diagram of pCold Ⅱvector shows the cloning site of HCV C gene insertion.</b>
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<b>Fig.3 The centrifuge pellets of bacteria shows the induced expression of fusion HCV C protein in E.coli</b>
 
<b>Fig.3 The centrifuge pellets of bacteria shows the induced expression of fusion HCV C protein in E.coli</b>
 
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<b>Fig.4 SDS-PAGE result showing the expression of HCV C-O173 and HCV C-O120, which were marked by GFP</b>
 
<b>Fig.4 SDS-PAGE result showing the expression of HCV C-O173 and HCV C-O120, which were marked by GFP</b>
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Latest revision as of 15:39, 17 October 2018

Our Parts

Our project is to develop a biosensor based on the aptamer target on the HCV core protein, using competition based rolling amplification. This device could be used for the detection of trace HCV virus in unpaid donation blood test and blood transfusion in the clinic, which could shorten the window period of HCV detection. In order to achieve enough HCV C protein expressed in Prokaryotes, we constructed some recombinant plasmids of HCV C gene, some of them are truncated or/and optimized, some of them are tagged with TEE and 6×His marker. We cloned all these modified HCV C genes into pSB1C3 plasmid backbone to produce new BioBrick parts, which are compatible with RFC 10. All those parts worked well, particularly BBa_K2608004, which was selected to use in our project.

HCV is a single strand RNA virus with a coat. Core antigen is the main protein of nucleocapsid. It is an important part of the virus particle. Core protein can control the virus itself, as well as the interactions of virus and host cells. After signal peptide was inserted to the 5’-UTR of HCV C gene, the expressed fusion protein can be secreted to the outside of the cell, with the ability of self-assembly into the virus particle. Ii is a very good tool for HCV research. All the part genes were inserted into the vector plasmid pColdⅡ, showed as below.

Fig.1 Map of pCold Ⅱvector shows TEE and His tag are inserted to the upstream of multiple cloning sites.

Fig.2 Diagram of pCold Ⅱvector shows the cloning site of HCV C gene insertion.

We constructed 10 HCV core protein parts with different length and different tag, even different sequences due to the codon optimization. All the part information are summarized in the table below.

In these parts, the BBa_K2608004 was the shortest one which contains 360bp, encoding 120aa without tagging other sequence. It was also optimized and expressed very well, which showed as following.

Fig.3 The centrifuge pellets of bacteria shows the induced expression of fusion HCV C protein in E.coli

Fig.4 SDS-PAGE result showing the expression of HCV C-O173 and HCV C-O120, which were marked by GFP

You can view our parts here:

<groupparts>iGEM18 JNFLS</groupparts>