Difference between revisions of "Part:BBa K2336026"
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<h1>'''Improvement'''</h1> | <h1>'''Improvement'''</h1> | ||
| − | To get the most efficient and effective capture effect,we designed to construct 12 different | + | 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.But 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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Revision as of 17:49, 31 October 2017
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 lanthanide binding tag(it is expressed as LBT in the following) 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 can 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 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 our system will be popular.
Characterization
This is one section for capture part.LBT1 can bind the lanthanide ions and be expressed on 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 let the LBT1 be expressed successfully,which means after we put the bacteria in the water,the lanthanide ions in the water would be bound and recycled effectively .
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 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.But 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.