Part:BBa_K2336030
NOTOC__ oprF-GS-FLAG-LBT7-GS-LBT7-GS-LBT7
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.
Contents
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 bound with the bacteria, and the concentration of the lanthanide ions in the water 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 production would be popular.
Characterization
This is one section for capture part.LBT1 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 LBT7 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.
DNA Gel Electrophoretic
To make sure that we get the target gene, we did the DNA gel electrophoretic to separate different gene. And here is the result.
Our target genes are 1488bp, and as the marker is DS5000, we could be sure that the bright bands in this picture are our target genes.
SDS-PAGE
After oprF-LBT7 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*LBT7 in test lane, which cannot be found in control lane. This result proves that the bacteria could express OprF-LBT7 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 to figure out whether the cell surface display system works or not.
Under fluorescence microscope, OprF-LBT7 can excitated a bright fluorescence at 488nm.
Improvement
To get the most efficient and effective capture effect, we designed to construct 12 different circuits with 12 different LBTs. Then we planned to test their binding force to get the best one. However we have not yet complete all the LBT construction in time. 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 compare different LBTs and they will be different in the ability of binding the lanthanide ions so that we can choose suitable LBTs in different conditions.
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