Difference between revisions of "Part:BBa K3593011"
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| − | <partinfo>BBa_K3593011 short</partinfo> | + | <partinfo>BBa_K3593011 short</partinfo><br> |
| − | < | + | This part codes the scFv that specifically binds to α-amanitin (a subtype of amatoxin) in vitro which derives from monoclonal antibody's Fvs[1]. The codon is optimized for E.coli for expression. This scFv has the highest affinity to α-amanitin, and lower affinity towards other subtypes in amatoxins. The scFv posed relatively good specificity with an IC50 of 77.48 ng/mL and IC15 of 1.91 ng/mL using ic-ELISA.<br> |
| − | + | This part cannot fully function on its own, so further modifications are needed.<br> | |
| − | + | See more at [https://parts.igem.org/Part:BBa_K3593012 BBa_K3593012]and [https://parts.igem.org/Part:BBa_K3593013 BBa_K3593013]. | |
| − | + | =Background= | |
| − | + | Amatoxins are chemicals present inside the genus Amanita and caused about 90% of mushroom poisoning. Being able to detect it before eating or in the field could possibly make a great decrease in people and animals who died because of poisonous mushrooms. Also being able to detect it in hospital can greatly help doctors in mushroom areas get correct information and do effective diagnosis to save the patient.<br> | |
| − | + | scFV is prepared using phage display to filter antibody fragments that have a high affinity towards antigens, which makes it become the smallest fragment of Ig molecule with function in binding antigens. So it is small, portable and has higher sensitivity compared with the original polyclonal antibody. And it can be produced in large scale in E.coli.<br> | |
| − | + | However, directly over-expression of scFv itself in E.coli will result in aggregation of peptides and insoluble inclusion bodies formed. So we have done further modification to this scFv.<br> | |
| − | + | See more at [https://parts.igem.org/Part:BBa_K3593013 BBa_K3593013]. | |
| − | + | ||
| − | + | =Sequence and features= | |
| − | + | <partinfo>BBa_K3593011 SequenceAndFeatures</partinfo> | |
| − | + | =Characterization of this scFv= | |
| − | + | ==Design== | |
| − | + | As our scFv's structure haven't been determined yet, we used a software called AbodyBuilder[2] to predict the structure of the scFv. In some of the CDR which determined the specificity and affinity of the scFv, the result of alignment is quite good, but others are not satisfying.<br> | |
| − | + | [[File:T--GreatBay_SCIE--scFv1.png|600px|thumb|center|Fig.1 The pridicted antibody structure]] | |
| − | + | [[File:T--GreatBay_SCIE--scFv2.png|600px|thumb|center|Fig.2 The pridicted antibody structure]] | |
| − | + | [[File:T--GreatBay_SCIE--scFv6.png|600px|thumb|center|Fig.3 The validation data of the pridicted model]] | |
| − | + | This result showed that we have the ability to predict and design our scFv and which means we can further develop our scFv to have a higher affinity and become more sensitive toward the toxin.<br> | |
| − | + | ==Experiment Data== | |
| − | + | ===The condition of best-producing scFv in BL21(DE3)-pET28b-pelB-scFv-HisTag is :=== | |
| − | + | 1. Inoculate strain from the plate into 4ml LB medium, add needed antibiotics ,cultivate at 37℃ 220RPM overnight. <br> 2. Attenuate the seed liquid into 20mL LB medium containing 100mM Glucose by the ration 1:50 to reduce leaked expression, add needed antibiotics. <br> 3. Cultivate the culture in 37℃, 220RPM until the OD600 reaches 0.9. <br> 4. 1500xg, 10 min, 25℃ centrifuge to collect cells in the medium <br> 5. Resuspend the pellet in same amount of 2YTS medium, add needed antibiotics. <br> 6. Add IPTG to final concentration 1mM.<br> 7. Cultivate the culture in room temperature, 200RPM for 24h. <br> 8. 5000xg, 10min, 25℃ to collect the cell. | |
| − | + | ===The formula of the two medium=== | |
| − | + | ||
| − | + | ||
<table border="1"> | <table border="1"> | ||
<tr> | <tr> | ||
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</tr> | </tr> | ||
</table> | </table> | ||
| − | + | ===Extraction of scFv in the cell pellet:=== | |
| − | + | We have used osmotic shock to extract the scFv in the periplasm, and then obtained the extract that can be used to conduct ELISA. The method we have used is as follows.<br> 1. Centrifuge, 5000xg 10 min at 4℃ to collect the cell.<br> 2. Resuspend the pellet in 10% original volume of 0.2 mol/L Tris–HCl, pH 8.0, 0.5 mmol/L EDTA, 0.5 mmol/L sucrose buffer. <br> 3. Incubate the mixture on ice for 30 min.<br> 4. Centrifuge, 12000xg 20min at 4℃, dispose the supernatant.<br> 5. Resuspend the pellet in 2% original volume of 5mM MgSO4 buffer, incubate at room temperature for 10 min.<br> 6. Centrifuge, 12000xg 20min at 4℃, collect the supernatant, and that is the Osmotic shock product. | |
| − | + | [[File:T--GreatBay_SCIE--osmotic_shock.jpg|600px|thumb|center|Fig.1 The SDS-PAGE showing the extracted periplasm protein and purified protein bands]] | |
| − | + | ===Purification of scFv=== | |
| − | + | After we have acquired the protein extract containing scFv, we need to purify it for further detection. Because we have added a His Tag to our recombinant protein, we can purify it through the IMAC. We have used Ni-NTA resin to purify the protein. However, we find that the affinity between the protein and resin is not very strong, so we change the washing buffer and remove the imdzole contained. <br> | |
| − | + | [[File:T--GreatBay_SCIE--purification2.jpg|600px|thumb|center|Fig.2 The SDS-PAGE showing the purified scFv with some other bands]] | |
| − | + | =References= | |
| − | + | 1. Zhang X, He K, Zhao R, Feng T, Wei D. Development of a Single Chain Variable Fragment Antibody and Application as Amatoxin Recognition Molecule in Surface Plasmon Resonance Sensors. Food Anal Methods. 2016. doi:10.1007/s12161-016-0509-3 <br> | |
| − | + | 2. Expression B. Bacterial Expression, Purification, and Characterization of Single-Chain Antibodies. :1035-1046. doi:10.1385/1-59259-169-8:1035 <br> | |
| − | + | 3. SAbPred: ABodyBuilder http://opig.stats.ox.ac.uk/webapps/newsabdab/sabpred/abodybuilder | |
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Latest revision as of 02:01, 28 October 2020
Single-Chain Fragment Variable (anti α-amatinin)
This part codes the scFv that specifically binds to α-amanitin (a subtype of amatoxin) in vitro which derives from monoclonal antibody's Fvs[1]. The codon is optimized for E.coli for expression. This scFv has the highest affinity to α-amanitin, and lower affinity towards other subtypes in amatoxins. The scFv posed relatively good specificity with an IC50 of 77.48 ng/mL and IC15 of 1.91 ng/mL using ic-ELISA.
This part cannot fully function on its own, so further modifications are needed.
See more at BBa_K3593012and BBa_K3593013.
Background
Amatoxins are chemicals present inside the genus Amanita and caused about 90% of mushroom poisoning. Being able to detect it before eating or in the field could possibly make a great decrease in people and animals who died because of poisonous mushrooms. Also being able to detect it in hospital can greatly help doctors in mushroom areas get correct information and do effective diagnosis to save the patient.
scFV is prepared using phage display to filter antibody fragments that have a high affinity towards antigens, which makes it become the smallest fragment of Ig molecule with function in binding antigens. So it is small, portable and has higher sensitivity compared with the original polyclonal antibody. And it can be produced in large scale in E.coli.
However, directly over-expression of scFv itself in E.coli will result in aggregation of peptides and insoluble inclusion bodies formed. So we have done further modification to this scFv.
See more at BBa_K3593013.
Sequence and features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 98
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Characterization of this scFv
Design
As our scFv's structure haven't been determined yet, we used a software called AbodyBuilder[2] to predict the structure of the scFv. In some of the CDR which determined the specificity and affinity of the scFv, the result of alignment is quite good, but others are not satisfying.
This result showed that we have the ability to predict and design our scFv and which means we can further develop our scFv to have a higher affinity and become more sensitive toward the toxin.
Experiment Data
The condition of best-producing scFv in BL21(DE3)-pET28b-pelB-scFv-HisTag is :
1. Inoculate strain from the plate into 4ml LB medium, add needed antibiotics ,cultivate at 37℃ 220RPM overnight.
2. Attenuate the seed liquid into 20mL LB medium containing 100mM Glucose by the ration 1:50 to reduce leaked expression, add needed antibiotics.
3. Cultivate the culture in 37℃, 220RPM until the OD600 reaches 0.9.
4. 1500xg, 10 min, 25℃ centrifuge to collect cells in the medium
5. Resuspend the pellet in same amount of 2YTS medium, add needed antibiotics.
6. Add IPTG to final concentration 1mM.
7. Cultivate the culture in room temperature, 200RPM for 24h.
8. 5000xg, 10min, 25℃ to collect the cell.
The formula of the two medium
| LB 100mM Gulcose formula/1L | 10 g of Bacto-Tryptone | 5 g of Bacto-Yeast extract | 18 g of Glucose | 5 g of NaCl | pH 7.5 |
| 2YTS medium/1L | 16 g of Bacto-Tryptone | 10 g of Bacto-Yeast extract | 137 g of Sucrose | 5 g of NaCl | pH 7.5 |
Extraction of scFv in the cell pellet:
We have used osmotic shock to extract the scFv in the periplasm, and then obtained the extract that can be used to conduct ELISA. The method we have used is as follows.
1. Centrifuge, 5000xg 10 min at 4℃ to collect the cell.
2. Resuspend the pellet in 10% original volume of 0.2 mol/L Tris–HCl, pH 8.0, 0.5 mmol/L EDTA, 0.5 mmol/L sucrose buffer.
3. Incubate the mixture on ice for 30 min.
4. Centrifuge, 12000xg 20min at 4℃, dispose the supernatant.
5. Resuspend the pellet in 2% original volume of 5mM MgSO4 buffer, incubate at room temperature for 10 min.
6. Centrifuge, 12000xg 20min at 4℃, collect the supernatant, and that is the Osmotic shock product.
Purification of scFv
After we have acquired the protein extract containing scFv, we need to purify it for further detection. Because we have added a His Tag to our recombinant protein, we can purify it through the IMAC. We have used Ni-NTA resin to purify the protein. However, we find that the affinity between the protein and resin is not very strong, so we change the washing buffer and remove the imdzole contained.
References
1. Zhang X, He K, Zhao R, Feng T, Wei D. Development of a Single Chain Variable Fragment Antibody and Application as Amatoxin Recognition Molecule in Surface Plasmon Resonance Sensors. Food Anal Methods. 2016. doi:10.1007/s12161-016-0509-3
2. Expression B. Bacterial Expression, Purification, and Characterization of Single-Chain Antibodies. :1035-1046. doi:10.1385/1-59259-169-8:1035
3. SAbPred: ABodyBuilder http://opig.stats.ox.ac.uk/webapps/newsabdab/sabpred/abodybuilder