Difference between revisions of "Part:BBa K3767002:Design"
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The structure of the 3-24 ScFv sequence was created by sourcing IgG ScFv fragments for both the VH and VL chains from Ghosh and Huber [[Part:BBa_K3767002#References|<sup>[1]</sup>]] and the 3-24 variant was selected as it was shown to have stronger binding to OpsA. Using SAbPred’s ABodyBuilder, we attempted to fold these chains together with the structures being based on orientation prediction, CDR modeling, and side chain prediction, and results are given a confidence score based on the root-mean square deviation threshold. The complementary determining region (CDR) anchor regions were determined by conducting a BLAST analysis of sequence homologies of the VH and VL sequences before the folding was predicted using ABodyBuilder. The confidence score of VH and VL are 0.89 and 0.99 respectively. | The structure of the 3-24 ScFv sequence was created by sourcing IgG ScFv fragments for both the VH and VL chains from Ghosh and Huber [[Part:BBa_K3767002#References|<sup>[1]</sup>]] and the 3-24 variant was selected as it was shown to have stronger binding to OpsA. Using SAbPred’s ABodyBuilder, we attempted to fold these chains together with the structures being based on orientation prediction, CDR modeling, and side chain prediction, and results are given a confidence score based on the root-mean square deviation threshold. The complementary determining region (CDR) anchor regions were determined by conducting a BLAST analysis of sequence homologies of the VH and VL sequences before the folding was predicted using ABodyBuilder. The confidence score of VH and VL are 0.89 and 0.99 respectively. | ||
| + | [[File:BLAST Scores ScFv.png|thumb|center|600px|Figure 5. Folding confidence model obtained for VH and VL confidence values of 0.89 and 0.99 using ABodyBuilder.]] | ||
Revision as of 01:10, 30 June 2021
Contents
Design
Once we were confident that the 3-24 ScFv Sequence would theoretically bind to OspA, we used UCSF Chimera in combination with PyMol to model a fused protein consisting of the 3-24 ScFv linked via C-terminal to phoA and was linked via (GGGS)3 linkers as glycine linkers are very flexible and limit strain between each component.
Considerations
We had to consider which phoA substrate to use as we would like to maximize the amount of florescence visible to the naked eye once the ScFv bound to OspA. We opted with BCIP as it is an improved version of phoA optimized for E.Coli with 40x catalytic activity, thus will fluoresce a more distinct blue color.
We began our design from the BBa_K1216001 part registered by the iGEM13_ETH_Zurich team[3]. From the translated sequence of this part, we realized there were two stop codons at the beginning of the sequence which led us to question how reliable the sequence was. To test reliability, we ran a BLAST analysis of the BBa_K1216001 and ran a Seaview alignment on the top 50 most similar sequences in E. coli. Based on this alignment we noticed that BBa_K1216001 had a N-terminus overhang that was not similar to other sequences. This led us to research alkaline phosphatase expressed primarily in E. coli. From this research we found the sequence created by Le Du, et al. (x40)[4] which we then aligned with BBa_K1216001 to compare similarities. In the alignment we found that the x40 sequence did not contain an N-terminal overhang and had two key mutations of D153G and D330N. The mutation at position 153 is extremely relevant as it located within the active site and thus plays an essential role in binding. As stated by Le Du, et al. this residue stabilizes a magnesium ion which when mutated increases activity. We did further alignment analysis of their 3D structures and found that there were minimal differences in structure compared to BBa_K1216001. These analyses suggested to use that the new x40 sequences was an improvement on BBa_K1216001 and therefore we codon optimized the x40 sequences for expression in E. Coli using Benchling.
Source
The structure of the 3-24 ScFv sequence was created by sourcing IgG ScFv fragments for both the VH and VL chains from Ghosh and Huber [1] and the 3-24 variant was selected as it was shown to have stronger binding to OpsA. Using SAbPred’s ABodyBuilder, we attempted to fold these chains together with the structures being based on orientation prediction, CDR modeling, and side chain prediction, and results are given a confidence score based on the root-mean square deviation threshold. The complementary determining region (CDR) anchor regions were determined by conducting a BLAST analysis of sequence homologies of the VH and VL sequences before the folding was predicted using ABodyBuilder. The confidence score of VH and VL are 0.89 and 0.99 respectively.
References
1. Ghosh, S., and Huber, B. T. (2007) Clonal diversification in OspA-specific antibodies from peripheral circulation of a chronic Lyme arthritis patient. J. Immunol. Methods. 321, 121–134
2. Sharma,U., Pal,D., and Prasad,R. (2013) Alkaline Phospatase: An Overview, Indian J Clin Biochem, [Online] https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4062654/, (accessed June 12 2021)
3. Part:BBa K1216001 - parts.igem.org [online] https://parts.igem.org/Part:BBa_K1216001#References (Accessed June 12, 2021)
4. Du, M. H. L., Lamoure, C., Muller, B. H., Bulgakov, O. V., Lajeunesse, E., Ménez, A., and Boulain, J. C. (2002) Artificial evolution of an enzyme active site: Structural studies of three highly active mutants of Escherichia coli alkaline phosphatase. J. Mol. Biol. 316, 941–953