Part:BBa_K2336028
oprF-GS-FLAG-LBT4-GS-LBT4-GS-LBT4
The capture 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.
Usage and biology
LBT（lanthanide binding tag） is a kind of protein which can bind with the lanthanide ions.With the help of OprF,our bacteria could express the LBT on its cell membrane and bind the lanthanide ions in the water.So once we put our bacteria into the water,the lanthanide ions in the water would be combined with the bacteria and the concentration of the lanthanide ions would decrease.
In this way,no matter how large the water volume is,we can put our bacteria in the water and let the bacteria bind the targeted ions in the polluted water.We can even link three specific LBTs for different water bodies.And as the pollution of lanthanide ions is serious,we think that the using of our system will be popular.
Characterization
This is one section of our capture part.LBT4 can bind lanthanide ions and it can be expressed at the E.coli's cell membrane with the help of OprF.We set the 'FLAG' between oprF and LBT for fluorescence detection.In our experiment,we make the LBT4 be expressed successfully, which means that the lanthanide ions in the water would be bound and recycled effectively after we put the bacteria in the water.
SDS-PAGE
After oprF-LBT4 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*LBT4 in test lane, which cannot be found in control lane.This result proves that the bacteria could express OprF-LBT4 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-LBT4 excitated a bright fluorescence at 488nm.
Titration
After the induction by IPTG,we centrifuged the bacteria liquid and put 3ml bacteria liquid in each tube with 40ml TbCl3.After reacting for an hour in the shaker and centrifugation,we precipitated the supernatant with NaOH.We titrated the precipitate with HCl.
Obviously,OprF-3*LBT4 could bind the Tb3+ effectively and decrease the concentration of Tb3+ in the water which means that our design is feasible. The effect of OprF-LBT could be clearly found by our eyes.
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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