Difference between revisions of "Part:BBa K2609003"
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<center><img src="https://static.igem.org/mediawiki/parts/8/87/T--IISc-Bangalore--phage_infection_mech.jpg" width=80% style="border: 1px solid black;"></center> | <center><img src="https://static.igem.org/mediawiki/parts/8/87/T--IISc-Bangalore--phage_infection_mech.jpg" width=80% style="border: 1px solid black;"></center> | ||
| − | + | <br> | |
| + | <p><b>Note that the protein requires two phage chaperones - gp38 and gp57 for proper folding</b>. Hence, during purification for structural studies, it should always be co-expressed with these.</p> | ||
<h2>Usage</h2> | <h2>Usage</h2> | ||
| − | The 2018 IISc-Bangalore iGEM team used an in silico <a href="http://2018.igem.org/Team:IISc-Bangalore/PhageModifier">'PhageModifier'</a> pipeline to modify the native gp37 to have increased affinity for phosphoethanolamine. This part is the coding sequence of the second such modification with a predicted binding affinity of | + | The 2018 IISc-Bangalore iGEM team used an in silico <a href="http://2018.igem.org/Team:IISc-Bangalore/PhageModifier">'PhageModifier'</a> pipeline to modify the native gp37 to have increased affinity for phosphoethanolamine. This part is the coding sequence of the second such modification with a predicted binding affinity of -6.42 kcal/mol for phosphoethanolamine (compared to the -3.4 kcal/mol of the wildtype protein).</p> |
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<p>The composite part containing gp37-M2 under a T7 promoter (with a 6xHis Tag followed by an enterokinase site on the N-term) was transformed into BL21 (DE3) and was induced at an OD of 0.6 (250uM IPTG) at 25°C for 16hrs. The cell pellet was run on a SDS-PAGE gel to check for expression of the protein.</p> | <p>The composite part containing gp37-M2 under a T7 promoter (with a 6xHis Tag followed by an enterokinase site on the N-term) was transformed into BL21 (DE3) and was induced at an OD of 0.6 (250uM IPTG) at 25°C for 16hrs. The cell pellet was run on a SDS-PAGE gel to check for expression of the protein.</p> | ||
| − | <p> | + | <p>A band was seen in the induced culture between 32kDa and 46kDa, <b>42kDa</b> being the expected size of the complete protein.</p> |
Latest revision as of 23:38, 17 October 2018
T4 bacteriophage long tail fiber protein gp37 (Modification II)
Coding sequence of the long tail fiber protein from T4 bacteriophage. The sequence has been modified in-silico for better binding to phosphoethanolamine near the receptor binding end of the protein. This is the second such modification in our series of four modifications.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 424
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
Biology
The T4 bacteriophage uses its long tail fiber to recognize and bind to its receptor OmpC on the surface of E. coli cells, its cognate host. The binding is mediated by non-covalent interactions that leads to the docking of the tip of the tail fiber (protein gp37) with OmpC in an extremely stable fashion.
This part is the coding sequence of a modified version of gp37 with the following amino acid changes
| Amino acid number | Original residue | Modified residue |
|---|---|---|
| 933 | I | D |
| 934 | E | D |
| 936 | W | Y |
| 944 | N | W |
| 945 | K | N |
| 946 | M | E |
| 959 | N | D |
| 960 | T | E |
| 961 | N | D |
The free movement of the rest of the phage body against the fixed tail leads to a confirmation change in the baseplate. This confirmation change pulls the entire phage to the cell surface followed by subsequent ejection of the phage DNA into the host[1]. The final injection happens with the aid of a "tail tube" that embeds itself into the outer membrane of the host and the mechanism is well conserved across multiple hosts because of similar membrane structures. The first interaction with the cell surface, i.e. the long tail fiber binding, is what determines the specificity of the phage to the host cell[2]. Modification of the tip of the tail fiber hence allows for a switching of the receptor that is used by the phage to infect the cell.
Note that the protein requires two phage chaperones - gp38 and gp57 for proper folding. Hence, during purification for structural studies, it should always be co-expressed with these.
Usage
The 2018 IISc-Bangalore iGEM team used an in silico 'PhageModifier' pipeline to modify the native gp37 to have increased affinity for phosphoethanolamine. This part is the coding sequence of the second such modification with a predicted binding affinity of -6.42 kcal/mol for phosphoethanolamine (compared to the -3.4 kcal/mol of the wildtype protein).Characterization
Expression with BBa_K2609013
The composite part containing gp37-M2 under a T7 promoter (with a 6xHis Tag followed by an enterokinase site on the N-term) was transformed into BL21 (DE3) and was induced at an OD of 0.6 (250uM IPTG) at 25°C for 16hrs. The cell pellet was run on a SDS-PAGE gel to check for expression of the protein.
A band was seen in the induced culture between 32kDa and 46kDa, 42kDa being the expected size of the complete protein.
References
[1] Leiman, Petr G., et al. "Three-dimensional rearrangement of proteins in the tail of bacteriophage T4 on infection of its host." Cell 118.4 (2004): 419-429.
[2] Yoichi, Masatoshi, et al. "Alteration of tail fiber protein gp38 enables T2 phage to infect Escherichia coli O157: H7." Journal of biotechnology 115.1 (2005): 101-107.
[3] Rakhuba, D. V., et al. "Bacteriophage receptors, mechanisms of phage adsorption and penetration into host cell." Pol. J. Microbiol 59.3 (2010): 145-155.
[4] Bartual, Sergio G., et al. "Structure of the bacteriophage T4 long tail fiber receptor-binding tip." Proceedings of the National Academy of Sciences 107.47 (2010): 20287-20292.