Team:Minnesota/Design
Team:Minnesota
#Minnesota 2018
Design:
Four engineered plasmids are constructed in our project.
the first plasmids (LacI-glnA-GFP)is constructed from pSB1C3 plasmid backbone consisting
LacI protein coding region, LacI binding site, glnA coding region and GFP. LacI protein coding region overexpression of LacI to ensure inhibition of LacI promoter
LacI binding site: controlled promoter region that only operates when LacI is released from IPTG binding
glnA coding region: expression of glutamine synthetase
GFP: Green fluorescent protein marker for characterizing the expression level of glnA
Other three plasmids are very similar in their sequence, it contains a full length Mer operon consists of MerR (mercuric regulatory protein),
transporter protein MerP and MerT, Mercury reductase MerA, and followed by glnA and GFP coding sequence.
The function of MerR is very similar to LacI, it binds to the promoter region when no ionic mercury is present and inhibits transcription.
When ionic mercury is present, it bind to MerR followed by MerR release.
The only difference between these three plasmids are the connecting sequence between glnA and Mer operon,
since we want the MerR (mercuric regulatory protein) to both control the transcription of Mer operon and glnA. Therefore, we have employed different binding sequences sources from different literature.
This way, we could test out which binding site is most efficient for our need. The bonding sequence is sourced from the sequence
in different Mer operon that are from different species (Tn501 and pDU1358) connecting MerR and MerP.
Functionally wise, glnA, GFP and merA will all be expressed when ionic mercury is present. glnA will support bacteria growth and merA reducing ionic mercury;
when the environmental mercury has all been exhausted, glnA synthesis will stop due to the inhibition of transcription and bacteria will eventually die off.
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