Team:Hong Kong HKUST/pneumosensor/future work

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<h2>Pneumosensor 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>
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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  
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with the detection and regulation modules by releasing specific bacteriophage lytic enzymes, Cpl-1 and Pal.
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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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<h5>Fig ? . The lysis Module.</h5>
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<p> The enzymes are tagged with osmY (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>.
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<u>References</u>
 
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J.M. Loeffler et al &quot;Rapid Killing of <i>Streptococcus pneumoniae</i> with a Bacteriophage Cell Wall Hydrolase&quot; 2001
 
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J.M. Loeffler et al &quot;Phage Lytic Enzyme Cpl-1 as a Novel Antimicrobial for Pneumococcal Bacteremia&quot; 2003
 
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J.M. Loeffler et al &quot;Synergistic Lethal Effect of a Combination of Phage Lytic Enzymes with Different Activities on Penicillin-Sensitive
 
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and -Resistant <i>Streptococcus pneumoniae</i> Strains&quot; 2003
 
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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
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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
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<div class='content_1'><h3><i>ComX</i>, PcelA, Phelicase Future Work </h3>
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<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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1. Put inducible promoter upstream of RBS (BBa_B0034), comX 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 comX protein expression and characterize combox promoters (PcelA and Phelicase) on different level of comX concentration.
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<u>References</u>
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2. Further characterization of combox promoters (PcelA and Phelicase) by making a 3-D Graph for time, comX concentration, and Fluorescence expression.
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J.M. Loeffler et al &quot;Rapid Killing of <i>Streptococcus pneumoniae</i> with a Bacteriophage Cell Wall Hydrolase&quot; 2001
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3. Characterizing combox promoters (PcelA and Phelicase) specificity by introducing other proteins with similar structure as comX protein.
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J.M. Loeffler et al &quot;Phage Lytic Enzyme Cpl-1 as a Novel Antimicrobial for Pneumococcal Bacteremia&quot; 2003
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4. Continue comW construction, ligate with terminator (BBa_B0015), and introduce it to E.coli DH10B strain.
 
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5. Characterization of comW by measuring the amount of comX protein with and without comW protein. (The function of comW protein is to protect comX protein from being degraded by ClpXP degradation enzyme)
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J.M. Loeffler et al &quot;Synergistic Lethal Effect of a Combination of Phage Lytic Enzymes with Different Activities on Penicillin-Sensitive
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and -Resistant <i>Streptococcus pneumoniae</i> Strains&quot; 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.


Amidase (PAL)- cleaves the peptidoglycan between N-acetylmuramic acid and L-alanine

Lysozyme (CPl-1)- cleaves the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine

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.

Moreover,ComW is a protein that function to protect σx from degradation. It is necessary to have ComW protein as it could increase the amount of σx to regulate PcelA and PcomFA 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-comW to a double terminator (BBa_B0015), and introduce it to E.coli DH10B strain. Then, characterization of comW could be performed by measuring the amount of σx with and without ComW protein.




References

J.M. Loeffler et al "Rapid Killing of Streptococcus pneumoniae with a Bacteriophage Cell Wall Hydrolase" 2001

J.M. Loeffler et al "Phage Lytic Enzyme Cpl-1 as a Novel Antimicrobial for Pneumococcal Bacteremia" 2003

J.M. Loeffler et al "Synergistic Lethal Effect of a Combination of Phage Lytic Enzymes with Different Activities on Penicillin-Sensitive and -Resistant Streptococcus pneumoniae Strains" 2003


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