Difference between revisions of "Part:BBa K3767001:Design"
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We began our design from the BBa_K1216001 part registered by the iGEM13_ETH_Zurich team[[Part:BBa_K3767001#References|<sup>[2]</sup>]] . From the translated sequence of this part, we realized there were two stop codons at the begin 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.  
 
We began our design from the BBa_K1216001 part registered by the iGEM13_ETH_Zurich team[[Part:BBa_K3767001#References|<sup>[2]</sup>]] . From the translated sequence of this part, we realized there were two stop codons at the begin 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.  
  
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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)[[Part:BBa_K3767001#References|<sup>[3]</sup>]]  which we then aligned with BBa_K1216001 to compare similarities. In the alignment we found that the x40 sequence did not contain a 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.
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[[File:BBa K3767001 Seaview alignment N-term overhang.png|thumb|center|800px|<b>Figure 1. N-terminal alignment of BBa K1216001 & BBa_J61032 with wild type, x17, and x40 alkaline phosphatase variants.</b>]] [[File:BBa K3767001 Seaview alignment of 153 mutation.png|thumb|center|800px|<b>Figure 2. Alignment of BBa K1216001 & BBa_J61032 with wild type, x17, and x40 alkaline phosphatase variants at mutated residue 153.</b>]] [[File:BBa K3767001 Seaview alignment of 330 mutation.png|thumb|center|800px|<b>Figure 3. Alignment of BBa K1216001 & BBa_J61032 with wild type, x17, and x40 alkaline phosphatase variants at mutated residue 330.</b>]]
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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)[[Part:BBa_K3767001#References|<sup>[3]</sup>]]  which we then aligned with BBa_K1216001 to compare similarities. In the alignment we found that the x40 sequence did not contain a 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. [[File:BBa K3767001 3D model alignment with BBa K1216001.png|thumb|center|800px|<b>Figure 4. 3D PDB structure alignment of x40 Alkaline phosphatase and BBa K1216001. BBa K1216001 and x40 Alkaline Phosphatase shwon in green and cyan respectively.</b>]]
  
  

Revision as of 17:07, 11 June 2021


Alkaline Phosphatase optimized for E. Coli w/ 40x catalytic activity


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

We began our design from the BBa_K1216001 part registered by the iGEM13_ETH_Zurich team[2] . From the translated sequence of this part, we realized there were two stop codons at the begin 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.

Figure 1. N-terminal alignment of BBa K1216001 & BBa_J61032 with wild type, x17, and x40 alkaline phosphatase variants.
Figure 2. Alignment of BBa K1216001 & BBa_J61032 with wild type, x17, and x40 alkaline phosphatase variants at mutated residue 153.
Figure 3. Alignment of BBa K1216001 & BBa_J61032 with wild type, x17, and x40 alkaline phosphatase variants at mutated residue 330.
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)[3] which we then aligned with BBa_K1216001 to compare similarities. In the alignment we found that the x40 sequence did not contain a 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.
Figure 4. 3D PDB structure alignment of x40 Alkaline phosphatase and BBa K1216001. BBa K1216001 and x40 Alkaline Phosphatase shwon in green and cyan respectively.


Source

PDB - https://www.rcsb.org/structure/1KH7?fbclid=IwAR0FDt9tFJP2mdON-9o4iJdtLiFRopQAhq2NDYREQO1OFwd9JBkx7ZSIwiU

References


1. Lowe, D., Sanvictores, T., and John, S. (2021) Alkaline Phosphatase, StatPearls Publishing, [online] http://www.ncbi.nlm.nih.gov/pubmed/29083622 (Accessed June 8, 2021)
2. Part:BBa K1216001 - parts.igem.org [online] https://parts.igem.org/Part:BBa_K1216001#References (Accessed June 8, 2021)
3. 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