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Team:Hong Kong HKUST/pneumosensor/future work
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| - | <h2>Future Work</h2> | + | <h2>Pneumosensor Future Work</h2> |
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<div class='content_1'><h3> Lysis Module </h3> | <div class='content_1'><h3> Lysis Module </h3> | ||
<table class="content_table" align= "center" > | <table class="content_table" align= "center" > | ||
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<p> | <p> | ||
Our team designed but was unable to test this module due to the limitation of not being able to work directly with <i>Streptococcus | Our team designed but was unable to test this module due to the limitation of not being able to work directly with <i>Streptococcus | ||
Pneumoniae</i> (Biosafety level 2) in our lab. This module proposes to kill <i>Streptococcus Pneumoniae</i> upon detection when coupled | Pneumoniae</i> (Biosafety level 2) in our lab. This module proposes to kill <i>Streptococcus Pneumoniae</i> upon detection when coupled | ||
| - | with the detection and | + | with the detection and modules by releasing specific bacteriophage lytic enzymes, Cpl-1 and Pal. |
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Amidase (PAL)- cleaves the peptidoglycan between N-acetylmuramic acid and L-alanine | Amidase (PAL)- cleaves the peptidoglycan between N-acetylmuramic acid and L-alanine | ||
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| - | + | <p> The enzymes are tagged with osmY (<a href="http://parts.igem.org/Part:BBa_K892008?title=Part:BBa_K892008">BBa_K892008</a>, Washington 2012) via a linker to be exported out of <i>Escherichia coli</i>. Both enzymes have | |
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very different N-terminal catalytic sites and share a similar C-terminal cell wall attachment site, which binds to choline in both cases. | very different N-terminal catalytic sites and share a similar C-terminal cell wall attachment site, which binds to choline in both cases. | ||
Cleavage with either of these enzymes results in a weakening in the cell wall, which leads to the externalization of the cytoplasmic | Cleavage with either of these enzymes results in a weakening in the cell wall, which leads to the externalization of the cytoplasmic | ||
membrane and ultimate lysis of <i>S. pneumoniae</i>. | membrane and ultimate lysis of <i>S. pneumoniae</i>. | ||
</p> | </p> | ||
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| + | <div class='content_1'><h3>P<sub>comCDE</sub></h3> | ||
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| + | <p>P<sub>comCDE</sub> is a promoter induced by phosphorylated ComE, which can be replaced by a phosphorylmimetic mutant (ComE<sup>D58E</sup>) in experiment. We could not | ||
| + | manage to verify the sequence of our ligated product, P<sub>comCDE</sub>- BBa_E0240. Therefore, in the future, we hope to continue the verification process, and complete our | ||
| + | construct.</p> | ||
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| + | <!-- one row of content , two column one picture left--> | ||
| + | <div class='content_1'><h3>σ<sup>x</sup>, P<sub>celA</sub>, P<sub>comFA</sub> </h3> | ||
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| + | <p> P<sub>celA</sub> and P<sub>comFA</sub> promoters are promoter that is regulated by σ<sup>x</sup>. We have proven that in the presence of σ<sup>x</sup>, P<sub>celA</sub> and P<sub>comFA</sub> could express GFP. However, we did not manage to characterize the GFP expression of P<sub>celA</sub> and P<sub>comFA</sub> in different concentration of σ<sup>x</sup>. So, a possible future work is to put inducible promoter upstream of RBS (BBa_B0034), σ<sup>x</sup> gene, and terminator (BBa_B0015). An example of inducible promoter is BBa_I0500. Hence, by putting an inducible promoter, we can tune the level of σ<sup>x</sup> expression and characterize Com-Box promoters (P<sub>celA</sub> and P<sub>comFA</sub>) on different level of σ<sup>x</sup> concentration. | ||
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| + | Moreover,ComW is a protein that function to protect σ<sup>x</sup> from degradation. It is necessary to have ComW protein as it could increase the amount of σ<sup>x</sup> to regulate P<sub>celA</sub> and P<sub>comFA</sub> promoters. However, due to the time constrains, we were unable to finish the construct of ComW generator. Hence, possible future work would be continuing ComW generator construct by ligating BBa_K880005-<i>comW</i> to a double terminator (BBa_B0015), and introduce it to <i>E.coli</i> DH10B strain. Then, characterization of <i>comW</i> could be performed by measuring the amount of σ<sup>x</sup> with and without ComW protein. | ||
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<div class= "content_area_two_row"> | <div class= "content_area_two_row"> | ||
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<u>References</u> | <u>References</u> | ||
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and -Resistant <i>Streptococcus pneumoniae</i> Strains" 2003 | and -Resistant <i>Streptococcus pneumoniae</i> Strains" 2003 | ||
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Latest revision as of 15:44, 14 November 2014
Pneumosensor Future Work
Lysis Module
|
Our team designed but was unable to test this module due to the limitation of not being able to work directly with Streptococcus Pneumoniae (Biosafety level 2) in our lab. This module proposes to kill Streptococcus Pneumoniae upon detection when coupled with the detection and modules by releasing specific bacteriophage lytic enzymes, Cpl-1 and Pal.
The enzymes are tagged with osmY (BBa_K892008, Washington 2012) via a linker to be exported out of Escherichia coli. Both enzymes have very different N-terminal catalytic sites and share a similar C-terminal cell wall attachment site, which binds to choline in both cases. Cleavage with either of these enzymes results in a weakening in the cell wall, which leads to the externalization of the cytoplasmic membrane and ultimate lysis of S. pneumoniae. |
PcomCDE
|
PcomCDE is a promoter induced by phosphorylated ComE, which can be replaced by a phosphorylmimetic mutant (ComED58E) in experiment. We could not manage to verify the sequence of our ligated product, PcomCDE- BBa_E0240. Therefore, in the future, we hope to continue the verification process, and complete our construct. |
σx, PcelA, PcomFA
|
PcelA and PcomFA promoters are promoter that is regulated by σx. We have proven that in the presence of σx, PcelA and PcomFA could express GFP. However, we did not manage to characterize the GFP expression of PcelA and PcomFA in different concentration of σx. So, a possible future work is to put inducible promoter upstream of RBS (BBa_B0034), σx gene, and terminator (BBa_B0015). An example of inducible promoter is BBa_I0500. Hence, by putting an inducible promoter, we can tune the level of σx expression and characterize Com-Box promoters (PcelA and PcomFA) on different level of σx concentration.
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