Team:Tsinghua/Parts
Bronze
Characterization of BBa_K1033932
This part is a pink chromogenic protein validated in E. coli. In an attempt to find alternative readout for our biosensor system, we constructed this part into
Silver
A. pGalS promoter of S. cerevisiae BBa_K2247000
This part is a Gal promoter from the yeast S. cerevisiae, modified from the endogenous Gal1 promoter. Endogenous Gal1 promoter contains upstream activating sequence (UAS), where lies the binding sites of the yeast Gal4 activator. Similar to pGal1, the expression from pGalS can be induced by Gal4 protein, or simply the addition of galactose, with a higher strength than pGal1. This promoter can serve as an positively regulated promoter in the S. cerevisiae system, or drive the expression of the reporter gene in any standard version of yeast-two-hybrid system. In our system, this promoter coupled the sensor and the report part, driving the expression of reporter genes when Gal4 AD and BD domains, each fused with an anti-aflatoxin B1 antibody, associated in the presence of aflatoxin B1.
B. HXT2 glucose transpoter from S. cerevisiae BBa_K2247002
High-affinity glucose transporter 2 (HXT2) is a member of the HXT transmembrane glucose transporter family of S. cerevisiae. HXT2 is localized to the plasma membrane, and has glucose, fructose, mannose and pentose transmembrane transporter activity. We expressed HXT2 in HXT-related genes knockout yeast (Strain JDYY001, modified from Strain EBY.VW4000 that is kindly provided by Dr. Junbiao Dai's Group from Tsinghua University), so that the decrease of glucose level in media can report the level of gene expression induced by aflatoxin, which we aimed to detect. In this way, we innovatively used glucometer as a method of readout, which can facilitate the household use of our system.
D. HXT5 glucose transpoter from S. cerevisiae BBa_K2247004
High-affinity glucose transporter 5 (HXT5) is a member of the HXT transmembrane glucose transporter family of S. cerevisiae, similar to HXT2. HXT5 is localized to the plasma membrane, and has glucose, fructose, mannose and pentose transmembrane transporter activity.
F. Single-chain variable fragment 1 of anti-Aflatoxin B1 antibody antiAFB1-ScFv1 BBa_K2247006
This part is a single-chain variable fragment of an anti-Aflatoxin B1 antibody, conferring the binding affinity to aflatoxin B1. Aflatoxin B1 is a major type of aflatoxin contaminating our everyday foods, and is a strong carcinogen to human. This part is a key element in our aflatoxin biosensor system.
G. Single-chain variable fragment 2 of anti-Aflatoxin B1 antibody (antiAFB1-ScFv2) BBa_K2247007
This part is a single-chain variable fragment of another anti-Aflatoxin B1 antibody, conferring the binding affinity to aflatoxin B1. Aflatoxin B1 is a major type of aflatoxin contaminating our everyday foods, and is a strong carcinogen to human. This part is a key element in our aflatoxin biosensor system.
H. AD-ScFv1 BBa_K2247008
This part is antiAFB1-ScFv1 (BBa_K2247006) fused with an N terminal yeast Gal4 transcription activation (AD) domain. This part, together with BD-ScFv2 (BBa_K2247009), 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.
I. BD-ScFv2 BBa_K2247009
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.
Gold
pGalS-GFP BBa_K2247001
This part encodes a GFP gene (BBa_K895006) under the pGalS yeast promoter, used to test the Gal4-dependent inducible expression from pGalS promoter (for detail, see BBa_K2247000). We have made a significant improvement of the original part, by validating that this GFP protein works well in the S. cerevisiae (baking yeast) chassis, and adding a yeast inducible promoter pGalS (BBa_K2247000) in front of this part. The new part we constructed is BBa_K2247001. By making this improvement, we achieved inducible expression of GFP by galactose or by yeast Gal4 protein in yeast cells, and this new composite part can also serve to validate and characterize the function of the pGalS promoter. In future projects applying the yeast-two-hybrid or yeast-one-hybrid system, this new part can serve as a basic and standard reporter plasmid. For experimental validation of the new part, please see the main page of BBa_K2247001.
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