Difference between revisions of "Team:CIEI-BJ/Parts"

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<div class="second-level" id="a2" >Parts</div>
 
<div class="second-level" id="a2" >Parts</div>
 
<div class="third-level" >BBa_K2631000: ADTZ Enzyme</div>
 
<div class="third-level" >BBa_K2631000: ADTZ Enzyme</div>
−
<p class="my-content" >The construct contains the coding sequence of ADTZ. ADTZ is also called aflatoxin oxidase (AFO), is the first enzyme identified to be able to degrade AFB1(Xu, T et al, 2017). It can be isolated from intracellular extracts and show a strong affinity with AFB1. As a potential degradation enzyme candidate in our project. In order to express soluble ADTZ in <i>E.coli<i>, the DNA coding ADTZ was inserted into the vector pMAL-c5x to produce the fusion protein of maltose-bindi protein (MBP) and ADTZ.</p>
+
<p class="my-content" >The construct contains the coding sequence of ADTZ. ADTZ is also called aflatoxin oxidase (AFO), is the first enzyme identified to be able to degrade AFB1(Xu, T et al, 2017). It can be isolated from intracellular extracts and show a strong affinity with AFB1. As a potential degradation enzyme candidate in our project. In order to express soluble ADTZ in <i>E.coli</i>, the DNA coding ADTZ was inserted into the vector pMAL-c5x to produce the fusion protein of maltose-bindi protein (MBP) and ADTZ.</p>
 
<div class="third-level" >BBa_K2631001: BD-ScFv2-pGAL1-eYFP</div>
 
<div class="third-level" >BBa_K2631001: BD-ScFv2-pGAL1-eYFP</div>
 
<p class="my-content" >This construct base on the IGEM parts (BBa_K2247009) contains an anti-AFB1 single chain variable fragments (ScFv2) targeting different affinity sites of AFB1 were fused with DNA binding domain (BD) , and ADH1 terminator (transcription terminator for alcohol dehydrogenase 1). We put another ADH1 terminator, the GAL1 promoter (inducible promoter, regulated by Gal4 in yeast, is used as the promoter in a standard yeast-two-hybrid system) cloned from S. cerevisiae (Johnston M et al, 1984) and an eYFP (enhanced YFP mutated from Aequorea Victoria GFP protein) downstream from the ScFv2 in the BamHI site of the vector (Biteen J S et al, 2008). This part, together with AD-ScFv1 (BBa_K2247008), plays the central role in the AFB1 biosensor system. The presence of AFB1 would draw near the two ScFvs, thus enabling the association of Gal4 AD and BD domains, and activate the EYFP protein, and driving the expression of the reporter gene in the AH109 yeast strain (from Takara). This design is that expression of eYFP under the control of the GAL4 induced promoter it regulates would produce a feedback, making the construct more sensitive to the AFB1 in the environment. eYFP act as a signal of the system are working.</p>
 
<p class="my-content" >This construct base on the IGEM parts (BBa_K2247009) contains an anti-AFB1 single chain variable fragments (ScFv2) targeting different affinity sites of AFB1 were fused with DNA binding domain (BD) , and ADH1 terminator (transcription terminator for alcohol dehydrogenase 1). We put another ADH1 terminator, the GAL1 promoter (inducible promoter, regulated by Gal4 in yeast, is used as the promoter in a standard yeast-two-hybrid system) cloned from S. cerevisiae (Johnston M et al, 1984) and an eYFP (enhanced YFP mutated from Aequorea Victoria GFP protein) downstream from the ScFv2 in the BamHI site of the vector (Biteen J S et al, 2008). This part, together with AD-ScFv1 (BBa_K2247008), plays the central role in the AFB1 biosensor system. The presence of AFB1 would draw near the two ScFvs, thus enabling the association of Gal4 AD and BD domains, and activate the EYFP protein, and driving the expression of the reporter gene in the AH109 yeast strain (from Takara). This design is that expression of eYFP under the control of the GAL4 induced promoter it regulates would produce a feedback, making the construct more sensitive to the AFB1 in the environment. eYFP act as a signal of the system are working.</p>

Revision as of 02:33, 16 October 2018

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Achiviments
Parts
BBa_K2631000: ADTZ Enzyme

The construct contains the coding sequence of ADTZ. ADTZ is also called aflatoxin oxidase (AFO), is the first enzyme identified to be able to degrade AFB1(Xu, T et al, 2017). It can be isolated from intracellular extracts and show a strong affinity with AFB1. As a potential degradation enzyme candidate in our project. In order to express soluble ADTZ in E.coli, the DNA coding ADTZ was inserted into the vector pMAL-c5x to produce the fusion protein of maltose-bindi protein (MBP) and ADTZ.

BBa_K2631001: BD-ScFv2-pGAL1-eYFP

This construct base on the IGEM parts (BBa_K2247009) contains an anti-AFB1 single chain variable fragments (ScFv2) targeting different affinity sites of AFB1 were fused with DNA binding domain (BD) , and ADH1 terminator (transcription terminator for alcohol dehydrogenase 1). We put another ADH1 terminator, the GAL1 promoter (inducible promoter, regulated by Gal4 in yeast, is used as the promoter in a standard yeast-two-hybrid system) cloned from S. cerevisiae (Johnston M et al, 1984) and an eYFP (enhanced YFP mutated from Aequorea Victoria GFP protein) downstream from the ScFv2 in the BamHI site of the vector (Biteen J S et al, 2008). This part, together with AD-ScFv1 (BBa_K2247008), plays the central role in the AFB1 biosensor system. The presence of AFB1 would draw near the two ScFvs, thus enabling the association of Gal4 AD and BD domains, and activate the EYFP protein, and driving the expression of the reporter gene in the AH109 yeast strain (from Takara). This design is that expression of eYFP under the control of the GAL4 induced promoter it regulates would produce a feedback, making the construct more sensitive to the AFB1 in the environment. eYFP act as a signal of the system are working.

<groupparts>iGEM18 CIEI-BJ</groupparts>