Difference between revisions of "Part:BBa K2247009"

 
 
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<partinfo>BBa_K2247009 short</partinfo>
 
<partinfo>BBa_K2247009 short</partinfo>
  
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antiAFB1-ScFv2, fused with N terminal yeast Gal4 DNA binding (BD) domain  
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This part is antiAFB1-ScFv2 (BBa_K2247007) fused with an N terminal yeast Gal4 DNA binding (BD) domain.
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===Usage and Biology===
 
===Usage and Biology===
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This part is antiAFB1-ScFv2 (BBa_K2247007) fused with an N terminal yeast Gal4 DNA binding (BD) domain. This part, together with AD-ScFv1 (BBa_K2247008), plays the central role in our AFT biosensor system. The presence of AFT would draw near the two ScFvs, thus enabling the association of Gal4 AD and BD domains, and driving the expression of the reporter gene.
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===Characterization===
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[[File:AFT sensor fusion protein design.jpeg|500px]]
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=====Figure 1. The design of our Aflatoxin-sensing system=====
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This scheme shows the working principle of our aflatoxin (AFT) biosensor system. Two anti-AFB1 single chain variable fragments (ScFv) targeting different affinity sites of AFT were fused with Gal4 transcription activation domain (AD) and DNA binding domain (BD) respectively. In the presence of AFT B1, the two anti-AFTB1 ScFvs could be drawn near, enabling the association of Gal4 AD and BD, and thus driving the expression of genes under the Gal promoter.
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[[File:AFT sensor validation.jpeg|500px]]
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=====Figure 2. Experimental validation of our Aflatoxin-sensing system=====
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This diagram shows the experiment we designed to validate the function of our AFT biosensor system. Yeast strain JDY26 (acquired from Dr. Junbiao Dai's lab in Tsinghua Univ.) was used, which contained and His3 gene under the control of the endogenous Gal1 promoter. According to our design, the presence of AFT B1 should lead to the association of Gal4 AD and BD domains, and drive the expression of His3, enabling the yeast to grow in Sc-His media. The growth of yeast cells in Sc-His media (denoted by OD 600) was used as the readout.
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[[File:AFT yeast His3 growth.jpeg|600px]]
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=====Figure 3. Expression from pGal promoter can be induced in the presence of AFT.=====
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As described in Figure 1 and Figure 2, the yeast strain JDY26 (ade2-101 trp1-901 leu2-3.112 his3∆200 ura3-52 gal4∆ gal80∆ SPAL::URA3 LYS2::GAL1-HIS GAL2-ADE2 met2:GAL7-LacZ can1R) was transformed with two plasmids, one encoding Gal4 AD domain fused with antiAFB1-ScFv1, and the other encoding Gal4 BD domain fused with antiAFB1-ScFv2. The yeast cells were cultured in Sc-His media with no Aflatoxin B1 (control) or 100 ug/L (100 ppb) AFT B1. According to our design, the presence of AFT B1 should facilitate the association of Gal4 AD and BD, thus driving the expression of His3 protein under the endogenous Gal1 promoter, and enabling yeast growth in Sc-His media. The yeast growth (OD 600) was measured every 1 hour for 30 hours on end, and the growth curve clearly showed that, compared to control, the presence of AFT B1 significantly promoted the growth of the yeast. Therefore, we believe the two anti-AFB1 antibody single-chain variable fragments (ScFv1, BBa_K2247006, and ScFv2, BBa_K2247007), as well as the fusion proteins AD-ScFv1 (BBa_K2247008) and BD-ScFv2 (BBa_K2247009) function well as expected.
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Latest revision as of 18:45, 31 October 2017


BD-ScFv2

This part is antiAFB1-ScFv2 (BBa_K2247007) fused with an N terminal yeast Gal4 DNA binding (BD) domain.


Usage and Biology

This part is antiAFB1-ScFv2 (BBa_K2247007) fused with an N terminal yeast Gal4 DNA binding (BD) domain. This part, together with AD-ScFv1 (BBa_K2247008), plays the central role in our AFT biosensor system. The presence of AFT would draw near the two ScFvs, thus enabling the association of Gal4 AD and BD domains, and driving the expression of the reporter gene.


Characterization

AFT sensor fusion protein design.jpeg


Figure 1. The design of our Aflatoxin-sensing system

This scheme shows the working principle of our aflatoxin (AFT) biosensor system. Two anti-AFB1 single chain variable fragments (ScFv) targeting different affinity sites of AFT were fused with Gal4 transcription activation domain (AD) and DNA binding domain (BD) respectively. In the presence of AFT B1, the two anti-AFTB1 ScFvs could be drawn near, enabling the association of Gal4 AD and BD, and thus driving the expression of genes under the Gal promoter.


AFT sensor validation.jpeg


Figure 2. Experimental validation of our Aflatoxin-sensing system

This diagram shows the experiment we designed to validate the function of our AFT biosensor system. Yeast strain JDY26 (acquired from Dr. Junbiao Dai's lab in Tsinghua Univ.) was used, which contained and His3 gene under the control of the endogenous Gal1 promoter. According to our design, the presence of AFT B1 should lead to the association of Gal4 AD and BD domains, and drive the expression of His3, enabling the yeast to grow in Sc-His media. The growth of yeast cells in Sc-His media (denoted by OD 600) was used as the readout.


AFT yeast His3 growth.jpeg


Figure 3. Expression from pGal promoter can be induced in the presence of AFT.

As described in Figure 1 and Figure 2, the yeast strain JDY26 (ade2-101 trp1-901 leu2-3.112 his3∆200 ura3-52 gal4∆ gal80∆ SPAL::URA3 LYS2::GAL1-HIS GAL2-ADE2 met2:GAL7-LacZ can1R) was transformed with two plasmids, one encoding Gal4 AD domain fused with antiAFB1-ScFv1, and the other encoding Gal4 BD domain fused with antiAFB1-ScFv2. The yeast cells were cultured in Sc-His media with no Aflatoxin B1 (control) or 100 ug/L (100 ppb) AFT B1. According to our design, the presence of AFT B1 should facilitate the association of Gal4 AD and BD, thus driving the expression of His3 protein under the endogenous Gal1 promoter, and enabling yeast growth in Sc-His media. The yeast growth (OD 600) was measured every 1 hour for 30 hours on end, and the growth curve clearly showed that, compared to control, the presence of AFT B1 significantly promoted the growth of the yeast. Therefore, we believe the two anti-AFB1 antibody single-chain variable fragments (ScFv1, BBa_K2247006, and ScFv2, BBa_K2247007), as well as the fusion proteins AD-ScFv1 (BBa_K2247008) and BD-ScFv2 (BBa_K2247009) function well as expected.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 237
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 156