Team:NYMU-Taipei/project/2c

From 2014.igem.org

(Difference between revisions)
Line 2: Line 2:
<html>
<html>
   <div id="main-content">
   <div id="main-content">
-
       <h>Communication</h>
+
       <h>Completion-Antibiofilm</h>
       <div class='abstract'>
       <div class='abstract'>
<ul>
<ul>
-
<li>The part aims to combine the two S. mutans killing system.</li>
+
<li>The circuit produces enzymes to degrade biofilms of Streptococcus mutans.</li>
-
<li>The phage-infected S. mutans emit quorum-sensing signal will pass the message down to the E.coli terminus then trigger the S. mutans killing module of it.</li>
+
<li>The circuit will be turned on when the environment is suitable for development of oral biofilms, which is under pH 4.0~5.0.</li>
-
<li>luxR-luxI system embedded into S. mutans and E. coli respectively constructs the communicative links between the two different germs.</li>
+
<li>The signal protein yebF can help to transport the product enzyme from E.coli to its extracellular environment.</li>
-
<li>The part reaches effective control on the number of S. mutans in case of that the threshold point defined by S. mutans’ number is surpassed.</li>
+
</ul>
</ul>
       </div>
       </div>
Line 21: Line 20:
       <div id='2c-1' style='height:50px;'></div>
       <div id='2c-1' style='height:50px;'></div>
       <h1>Purpose</h1>
       <h1>Purpose</h1>
-
<p>Targeting at the tooth problems, we are absolutely not going to rely on only one shoot on it. Our team is seeking for combinations of remedies to reach certain synergistic effect on preventing oral cavity.</p>
+
      <p>Streptococcus Mutans will form biofilms, a polymeric conglomeration generally consists of extracellular DNA, proteins, and polysaccharides. Biofilm is known for causing dental plaque, tooth decay and gum infection. Also, biofilm make it hard for our engineered probiotics to capture and kill the streptococcus mutans inside our mouths. Nowadays, the most basic way of removing biofilm is by physical methods such as brushing one's teeth regularly. However, there are many people who are not able to do so themselves, so we wanted to innovate and create a biological method to get rid of biofilms with our engineered probiotics. Therefore, we are going to design a circuit that produce several enzymes to destroy biofilm structure.</p>
       <div id='2c-2' style='height:50px;'></div>
       <div id='2c-2' style='height:50px;'></div>
       <h1>Background</h1>
       <h1>Background</h1>
-
<p>The two main organisms we adapted in our project are E. coli and M102 bacteriophage, respectively. Our team comes up with an interesting thought that we want to let these two entirely different organisms collaborate, thus we decided to use one of the most commonly seen quorum sensing chemicals, AHL, N-Acyl-homoserine-lactone, as our indicator between phage and E.coli. Due to the idea of phage threshold control of the number of S. mutans in prevention of the second-dominant strain in the oral cavity, we envision a possibility to trigger another killing system to reach synergistic effect on diminishing S. mutans. If the threshold terminators were passed, the downstream signaling pathway would be switched on and contact with the engineered E.coli.</p>
+
      <p>Extracellular polymeric substance (EPS) is important in the formation of biofilm. The EPS matrix consists of polysaccharides, proteins and nucleic acids. The dense extracellular matrix of the biofilms and the outer layer of cells protect the interior of the community, and in some cases, even increase antibiotic resistance. Even though the biofilm seem to be robust, we found that enzymatic degradation is able to weaken the biofilm structure.</p><br>
 +
      <p>As mentioned before, we are going to use antibiofilm enzyme as our tool. According to 2012 INSA-Lyon and HIT-Harbin iGEM teams, we decided to use lysostaphin as our ideal enzyme. Lysostaphin has activities of 3 enzymes, including glycylglycine endopeptidase, endo-β-N-acetyl glucosamidase and N-acteyl muramyl-L-alanine amidase. Those enzymes can cleave the glycine–glycine bonds, which form cross links between glycopeptide chains in the cell wall peptidoglycan, and cleave the peptidoglycan of a backbone made up of alternating β-1,4 linked N-acetylglucosamine and N-acetylmuramic acid residues as well.</p>
       <div id='2c-3' style='height:50px;'></div>
       <div id='2c-3' style='height:50px;'></div>
       <h1>Design</h1>
       <h1>Design</h1>
-
<p>The circuit design of “communication module” could be divided into two parts, one in Streptococcus mutans, and the other in Escherichia coli.</p><br>
+
      <p>
-
<p><b>The signal producer: Streptococcus mutans.</b></p><br>
+
<b>Promoter (Asr promoter)</b><br>
-
<p>LuxI(BBa_C0061):<br>
+
The Asr promoter comes from Escherichia coli strain MG1655, and has the ability to be induced by external acidification (pH 4.0 ~ 5.0). The sequence of Asr promoter includes an open reading frame coding for a polypeptide consists of 111 amino acids. Besides, according to computer-assisted analysis, the predicted polypeptide also contains a typical signal sequence of 30 amino acids, and it might represent either a periplasmic protein or an outer membrane protein.<br><br>
-
Coding sequence lies after the threshold terminator of nlmc promoter. LuxI acts as a gene which would generate AHL-synthase that yielding the quorum-sensing signal AHL right after the threshold level of S.mutans is exceeded.</p><br>
+
<b>Signal protein (yebF with RBS)</b><br>
-
<p>Terminator(BBa_B0015):<br>
+
As described in the killing part, yebF is a transporting protein naturally secreted by Escherichia coli, and it can also be used in experimental strains which are rarely involved in protein secretion.<br><br>
-
Double terminator combined by terminator BBa_B0010 and BBa_B0012.</p><br>
+
<b>Lysostaphin producer (iGEM part BBa_K748002)</b><br>
-
<p><b>Signal receptor and killer module: Escherichia coli.</b></p><br>
+
Part BBa_K748002 comes from 2012 HIT-Harbin iGEM team, and it encodes lysostaphin. Lysostaphin is naturally secreted by Staphylococcus simulans. It is a zinc-containing metalloenzyme of 27 kDa, and has activities of three different enzymes, which are glycylglycine endopeptidase, endo-β-N-acetyl glucosamidase and N-acetyl-muramyl-L-alanine amidase. Glycylglycine endopeptidase hydrolyzes glycylglycine bonds in the polyglycine bridges that form cross links between glycopeptide chains in the cell wall peptidoglycan. As for endo-β-N-acetyl glucosamidase and N-acetyl-muramyl-L-alanine amidase, they can cleave the bonds between N-acetyl glucosamine, N-acetyl muramic acid and alanine respectively. Moreover, lysostaphin is able to lyse actively growing and non-dividing cells in biofilms rapidly, whereas most antibiotics works effectively only on actively dividing cells. Last but not least, lysostaphin has low toxic and cause nearly no side effect on human body. Therefore, we select it as our lytic enzyme against the biofilms of Streptococcus mutans.<br><br>
-
<p>Constitutive promoter + RBS:<br>
+
<b>Terminator (iGEM part BBa_B0015)</b><br>
-
Sequences acquired by primer PCR from the template BBa_K523013 acts as promoter and ribosome binging site.</p><br>
+
A double terminator composed of B0010 and B0012. It has used by many previous teams, and have strong terminating force.  
-
<p>LuxR(BBa_C0062):<br>
+
      </p>
-
Coding sequence for LuxR protein that would complex with the quorum-sensing signal AHL. and then turns into an activated form which will afterwards bind with the promoter pLuxR and triggers it.</p><br>
+
-
<p>Terminator(BBa_B0015):<br>
+
-
Double terminator combined by terminator BBa_B0010 and BBa_B0012.</p><br>
+
-
<p>pLuxR promoter(BBa_R0062):<br>
+
-
Coding sequence for an inducible promoter pLuxR that would open the downstream killer module of Escherichia coli targeting at the cell walls of Streptococcus mutans in order to cause cell death of it.</p>
+
       <div id='2c-4' style='height:50px;'></div>
       <div id='2c-4' style='height:50px;'></div>
       <h1>Functional Measurement</h1>
       <h1>Functional Measurement</h1>
-
<p>To make a proof of our circuit design, we construct the functional test as a four-stages program.<br>
+
      <p>
 +
For testing our circuit’s function, we designed a set of experiments.<br><br>
 +
<b>Stage one— Test the Asr promoter</b><br>
 +
Asr promoter(with RBS)+RFP+Terminator<br><br>
 +
Transform the circuit to Escherichia coli, and grow the modified E. coli on agar plates overnight.<br><br>
 +
<i>Steps:</i><br></p>
 +
<ol class='cont-ol'>
 +
<li>Pick both red and non-red colonies, and grow the E.coli in separate liquid cultures.</li>
 +
<li>Prepare 2 sets of 2 plates, one pH 4 and one pH 7. Spread the red strain liquid culture on one set of the ph4 and 7 plates, and repeat on the other set with the non-red strain.</li><li>Select the strain that expresses growth differences from the previous step. Prepare two of each liquid culture tubes for pH 3.5, 4, 4.5, 5, 5.5, 6, 6.5, and 7, and grow the selected strain in all tubes. Use the growth data from each tube to plot the precise effect of pH on asr promoter.</li>
 +
<li>Measure the OD value (RFP/bacterium number).</li>
 +
</ol>
<br>
<br>
-
<b>Stage one: Circuit construction check.</b><br>
+
<p>
-
After all the fragments of the circuit are linked, we need to test whether all the fragments are well-connected. Thus we design to insert red fluorescent protein gene (BBa_J06602) at the end of our coding sequence to ensure our whole circuit is constructed correctly.<br>
+
<b>Stage two— Test the signal protein yebF</b><br>
 +
(1) J23100+yebF(with RBS)+RFP+Terminator<br>
 +
(2) J23100+RFP+Terminator<br><br>
 +
Transform both circuits to Escherichia coli, and grow the modified E.coli liquid culture overnight.<br><br>
 +
<i>Steps:</i><br></p>
 +
<ol class='cont-ol'>
 +
<li>Adjust the two cultures to the approximate OD value.</li>
 +
<li>Centrifuge the cultures and extract the supernatants.</li>
 +
<li>If the YebF signal sequence does work, the E.coli supernatant containing first circuit should be red while the second circuit having normal culture color.</li>
 +
</ol>
<br>
<br>
-
<b>Stage two: AHL releasing test.</b><br>
+
<p>
-
Our main concern occurs that AHL is a gram-negative quorum sensing-signal yet we are going to let the engineered S.mutans which is actual a gram-positive bacteria to express and transmit AHL. In this stage, we are going to make a standard curve of the concentration of the commercialized AHL versus the absorption value first. Then transform the circuit containing LuxI into Streptococcus mutans. By extracting the supernatant of the culturing medium of the engineered S.mutans, and use spectrometer to measure the concentration of AHL compares to the standard curve made previously.<br>
+
<b>Stage three—Test the lysostaphin producer iGEM part BBa_K748002 with yebF</b><br>
-
<br>
+
(1) J23100-(RBS)yebF-lysostaphin-terminator<br>  
-
<b>Stage three: Killer module inducing test.</b><br>
+
(2) J23100-(RBS)yebF-terminator<br><br>
-
In this stage, we are adding different amount of commercialized AHL to the engineered Escherichia coli implanted with our constructed circuit. We need to co-culture the E.coli and the normal Streptococcus mutans without our constructed circuit in this functional test program. The anticipating result will be reflected by the decrease of the number of normal S.mutans if the killer module is successfully activated by the quorum-sensing signal AHL.<br>
+
<i>Steps:</i><br></p>
-
<br>
+
<ol class='cont-ol'>
-
<b>Stage four: Final test with all combinations of the constructed circuits.</b><br>
+
<li>Transform both circuits to Escherichia coli, and grow the modified E.coli liquid culture overnight. At the meantime, culture Streptococcus mutans biofilms, and add crystal violet to label the location of the biofilms.</li>
-
In this last stage we would put all the constructed circuits respectively into Escherichia coli and Streptococcus mutans. Co-culture of E.coli and S.mutans is also required in this testing program to make sure our expecting interaction between the two different engineered germs is conducted and well-functioned.
+
<li>Adjust the two cultures to the approximate OD value. </li>
-
</p>
+
<li>Centrifuge the cultures and extract the supernatants.</li>
 +
<li>Finally, add the solution into the liquid culture of Streptococcus mutans. Use confocal microscope to check the crystal violet stain. The amount of crystal violet stain can show if the biofilms are degraded or not.</li>
 +
</ol>
       <div id='2c-5' style='height:50px;'></div>
       <div id='2c-5' style='height:50px;'></div>
       <h1>Result</h1>
       <h1>Result</h1>
 +
      <p></p>
       <h1>Reference</h1>
       <h1>Reference</h1>
-
  </div>
+
      <p></p>
 +
  </div>
</html>
</html>
{{:Team:NYMU-Taipei/NYMU14_Footer}}
{{:Team:NYMU-Taipei/NYMU14_Footer}}

Revision as of 13:01, 7 October 2014

X
Next ⇒
⇐ Prev
...
Completion-Antibiofilm
  • The circuit produces enzymes to degrade biofilms of Streptococcus mutans.
  • The circuit will be turned on when the environment is suitable for development of oral biofilms, which is under pH 4.0~5.0.
  • The signal protein yebF can help to transport the product enzyme from E.coli to its extracellular environment.

purpose

background

design

Functional Measurement

result

Purpose

Streptococcus Mutans will form biofilms, a polymeric conglomeration generally consists of extracellular DNA, proteins, and polysaccharides. Biofilm is known for causing dental plaque, tooth decay and gum infection. Also, biofilm make it hard for our engineered probiotics to capture and kill the streptococcus mutans inside our mouths. Nowadays, the most basic way of removing biofilm is by physical methods such as brushing one's teeth regularly. However, there are many people who are not able to do so themselves, so we wanted to innovate and create a biological method to get rid of biofilms with our engineered probiotics. Therefore, we are going to design a circuit that produce several enzymes to destroy biofilm structure.

Background

Extracellular polymeric substance (EPS) is important in the formation of biofilm. The EPS matrix consists of polysaccharides, proteins and nucleic acids. The dense extracellular matrix of the biofilms and the outer layer of cells protect the interior of the community, and in some cases, even increase antibiotic resistance. Even though the biofilm seem to be robust, we found that enzymatic degradation is able to weaken the biofilm structure.


As mentioned before, we are going to use antibiofilm enzyme as our tool. According to 2012 INSA-Lyon and HIT-Harbin iGEM teams, we decided to use lysostaphin as our ideal enzyme. Lysostaphin has activities of 3 enzymes, including glycylglycine endopeptidase, endo-β-N-acetyl glucosamidase and N-acteyl muramyl-L-alanine amidase. Those enzymes can cleave the glycine–glycine bonds, which form cross links between glycopeptide chains in the cell wall peptidoglycan, and cleave the peptidoglycan of a backbone made up of alternating β-1,4 linked N-acetylglucosamine and N-acetylmuramic acid residues as well.

Design

Promoter (Asr promoter)
The Asr promoter comes from Escherichia coli strain MG1655, and has the ability to be induced by external acidification (pH 4.0 ~ 5.0). The sequence of Asr promoter includes an open reading frame coding for a polypeptide consists of 111 amino acids. Besides, according to computer-assisted analysis, the predicted polypeptide also contains a typical signal sequence of 30 amino acids, and it might represent either a periplasmic protein or an outer membrane protein.

Signal protein (yebF with RBS)
As described in the killing part, yebF is a transporting protein naturally secreted by Escherichia coli, and it can also be used in experimental strains which are rarely involved in protein secretion.

Lysostaphin producer (iGEM part BBa_K748002)
Part BBa_K748002 comes from 2012 HIT-Harbin iGEM team, and it encodes lysostaphin. Lysostaphin is naturally secreted by Staphylococcus simulans. It is a zinc-containing metalloenzyme of 27 kDa, and has activities of three different enzymes, which are glycylglycine endopeptidase, endo-β-N-acetyl glucosamidase and N-acetyl-muramyl-L-alanine amidase. Glycylglycine endopeptidase hydrolyzes glycylglycine bonds in the polyglycine bridges that form cross links between glycopeptide chains in the cell wall peptidoglycan. As for endo-β-N-acetyl glucosamidase and N-acetyl-muramyl-L-alanine amidase, they can cleave the bonds between N-acetyl glucosamine, N-acetyl muramic acid and alanine respectively. Moreover, lysostaphin is able to lyse actively growing and non-dividing cells in biofilms rapidly, whereas most antibiotics works effectively only on actively dividing cells. Last but not least, lysostaphin has low toxic and cause nearly no side effect on human body. Therefore, we select it as our lytic enzyme against the biofilms of Streptococcus mutans.

Terminator (iGEM part BBa_B0015)
A double terminator composed of B0010 and B0012. It has used by many previous teams, and have strong terminating force.

Functional Measurement

For testing our circuit’s function, we designed a set of experiments.

Stage one— Test the Asr promoter
Asr promoter(with RBS)+RFP+Terminator

Transform the circuit to Escherichia coli, and grow the modified E. coli on agar plates overnight.

Steps:

  1. Pick both red and non-red colonies, and grow the E.coli in separate liquid cultures.
  2. Prepare 2 sets of 2 plates, one pH 4 and one pH 7. Spread the red strain liquid culture on one set of the ph4 and 7 plates, and repeat on the other set with the non-red strain.
  3. Select the strain that expresses growth differences from the previous step. Prepare two of each liquid culture tubes for pH 3.5, 4, 4.5, 5, 5.5, 6, 6.5, and 7, and grow the selected strain in all tubes. Use the growth data from each tube to plot the precise effect of pH on asr promoter.
  4. Measure the OD value (RFP/bacterium number).

Stage two— Test the signal protein yebF
(1) J23100+yebF(with RBS)+RFP+Terminator
(2) J23100+RFP+Terminator

Transform both circuits to Escherichia coli, and grow the modified E.coli liquid culture overnight.

Steps:

  1. Adjust the two cultures to the approximate OD value.
  2. Centrifuge the cultures and extract the supernatants.
  3. If the YebF signal sequence does work, the E.coli supernatant containing first circuit should be red while the second circuit having normal culture color.

Stage three—Test the lysostaphin producer iGEM part BBa_K748002 with yebF
(1) J23100-(RBS)yebF-lysostaphin-terminator
(2) J23100-(RBS)yebF-terminator

Steps:

  1. Transform both circuits to Escherichia coli, and grow the modified E.coli liquid culture overnight. At the meantime, culture Streptococcus mutans biofilms, and add crystal violet to label the location of the biofilms.
  2. Adjust the two cultures to the approximate OD value.
  3. Centrifuge the cultures and extract the supernatants.
  4. Finally, add the solution into the liquid culture of Streptococcus mutans. Use confocal microscope to check the crystal violet stain. The amount of crystal violet stain can show if the biofilms are degraded or not.

Result

Reference

Retrieved from "http://2014.igem.org/Team:NYMU-Taipei/project/2c"