Difference between revisions of "Part:BBa K2609013"
 
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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>
 
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<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>
  
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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 first such modification with a predicted binding affinity of ____ kcal/mol for phosphoethanolamine (compared to the ___kcal/mol of the wildtype protein).</p>  
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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 first 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>  
  
  
 
<h2>Characterization</h2>
 
<h2>Characterization</h2>
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<figcaption><a href="https://parts.igem.org/Part:BBa_K2609007">BBa_K2609013</a> expressed in BL21 (DE3) at 25°C for 16 hrs. 1mL of the culture was resuspended in 100uL PBS and mixed with 100uL of 2xSDS Sample Buffer followed by boiling. 20uL of the final mix was loaded onto a SDS-PAGE gel.</figcaption>
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<h3>Expression with <a href="https://parts.igem.org/Part:BBa_K2609013">BBa_K2609013</a></h3>
 
<h3>Expression with <a href="https://parts.igem.org/Part:BBa_K2609013">BBa_K2609013</a></h3>
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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.
 
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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>
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<p>Bands were found near 25kDa as opposed to 44kDa which is the expected size of the protein. The production of the expected protein from this part is hence non-conclusive.</p>
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Latest revision as of 23:57, 17 October 2018


gp37 under T7 expression system - Mod II

Generator for the long tail fiber protein from T4 bacteriophage under the T7 expression system. The protein is fused to a 6xHis tag at the N-term through a enterokinase tag for easy purification. 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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1239
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 507
  • 1000
    COMPATIBLE 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
935 A D
944 N Y
945 K L
946 M S
959 N D
960 T E
961 N E

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 first 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

BBa_K2609013 expressed in BL21 (DE3) at 25°C for 16 hrs. 1mL of the culture was resuspended in 100uL PBS and mixed with 100uL of 2xSDS Sample Buffer followed by boiling. 20uL of the final mix was loaded onto a SDS-PAGE gel.

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.

Bands were found near 25kDa as opposed to 44kDa which is the expected size of the protein. The production of the expected protein from this part is hence non-conclusive.

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.