Part:BBa_K2336026
oprF-GS-FLAG-LBT1-GS-LBT1-GS-LBT1
The report part of our circuit. Oprf can be expressed as an anchor on the cell membrane to make sure the LBT can combine with terbium (Tb3 +) on the surface of the bacteria.
Characterization
This is a part for recycling.LBT1（lanthanide binding tag）can bind the lanthanide ions and it can be expressed at the cell membrane of E.coli with the help of oprF.We set the 'FLAG' between oprF and LBT for fluorescence detection.In our experiment,we let the LBT1 be expressed successfully which means that the lanthanide ions in the water would be bound and recycled effectively after we put the in the water.
SDS-PAGE
After oprF-LBT1 is expressed successfully,we centrifuged the bacteria liquid and separated different proteins by SDS-PAGE.
Figure shows an obvious ~31kDa protein bands of oprF-3*LBT1 in test lane, which cannot be found in control lane.This result proves that the bacteria could express oprF-LBT1 successfully.
Fluorescence Detection
After the induction by IPTG,we use DYKDDDDK Tag (9A3) Mouse mAb (Binds to same epitope as Sigma's Anti-FLAG M2 Antibody) as the primary antibody and the Anti-mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor 488 Conjugate) as the second antibody.
Under fluorescence microscope,oprF-LBT1 excitated a bright fluorescence.
Improvement
As we didn’t have enough time,we couldn’t let all the 12 LBT be expressed successfully(LBT2/5/9 are failed).After competition of this year,HUST-China will get these 3 LBTs to be expressed and improve the efficiency of expression.If we make it,we will link different LBTs together and they will be different in the ability of binding the lanthanide ions so that we can link suitable LBTs for each water body.
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