Team:Nanjing-China/Parts
A composite of hydrogen sensor full length sequence. The order of the elements is: HoxA-HoxB-HoxC-HoxJ-terminator-HoxP-EGFP. The sequence of HoxABCJP comes from Ralstonia eutropha H16 megaplasmid pHG1. The hole sequence acts as an hydrogen sensor.
SQR is a NAD(P)/FAD-dependent oxidoreductase, which oxidizes S2- to zero-valent S0. SqrR, encoded by sqrR, is a constitutively expressed suppressor protein of sqr promotor. RFP is the indicator. When there is no H2S in the environment, SqrR combines with the sqr promotor and keeps the expression of both the enzyme and RFP at a low level. While in the case where H2S exists, it will be oxidized by the small amount of expressed enzyme, and the product will interact with SqrR, forming a tri- or tetrasulfide cross-links linking C41 and C107 on the same subunit of SqrR. The change in the structure of SqrR makes it fall off from the sqr promotor and thus the expression of SQR and RFP turns on. The part can be used to detect hydrogen sulfide at a μmol level.
The sequence is composed of PfrmR, gene frmR, flag tag, PfrmAB, gene RFP.
When the pathway works, we can see that the E.coli turns to red with naked eyes at the presence of formaldehyde.
The fluorescence intensity of protein RFP and the depth of the color in naked eyes are both in positive correlation with the concentration of formaldehyde within the tolerable range.
Since we have constructed three composite parts for gas-regulated gene expression. We are competing for the Best Part Collection Prize this year. Each of them contains a coding sequence for a key protein interacting with one of the three kinds of gas molecules. Those key proteins, combined with or dissociated from gas molecules, act as transcriptional regulator to downstream genes. In order to make their function noticeable, we have included visible reporter genes in each part. Our part collection is not only suitable for visualized gas detection, but also good for other purposes as long as users want their pathways to be regulated by hydrogen, hydrogen sulfide or formaldehyde.
Address
Life Science Department
#163 Xianlin Blvd, Qixia District
Nanjing University
Nanjing, Jiangsu Province
P.R. of China
Zip: 210046
nanjing_china@163.com
Social media
Twitter button:
iGEM Nanjing-China@nanjing_china
