Team:Hong Kong HKU/project/cargo

From 2013.igem.org

(Difference between revisions)
(Created page with "{{:Team:Hong_Kong_HKU/templates/header_v2}} {{:Team:Hong_Kong_HKU/templates/floatingmenu/project}} <html> <body> <style type="text/css"> #contentpicture{ background-colo...")
 
(21 intermediate revisions not shown)
Line 4: Line 4:
<html>
<html>
 +
 +
<head>
 +
<script>
 +
$(window).scroll(function() {
 +
var Content = $(this).scrollTop();
 +
if (Content > 525) {
 +
$('.floating-menu').fadeIn();
 +
}else {
 +
$('.floating-menu').fadeOut();
 +
}
 +
});
 +
</script>
 +
</head>
 +
<body>
<body>
<style type="text/css">
<style type="text/css">
Line 28: Line 42:
}
}
</style>
</style>
-
<div id="wrapper">  
+
<div id="wrapper">
-
<div id="content1">
+
<image src="https://static.igem.org/mediawiki/2013/c/cf/Upload_panel_project_00000.jpg" height=auto width="960" />
-
<img src="https://static.igem.org/mediawiki/2013/d/d9/Leaflet_ecapsi.jpg" height="500" width=auto>
+
-
<p>
+
-
<font face="Arial" size="2">Human practice in the iGEM competition is about introducing synthetic biology to people through various ways.  This year, the HKU iGEM team wanted to arouse people’s interest towards this new field of science by organizing some talks for both high school students and undergraduates.  We also made an animated video about our project which is broadcasted on YouTube for public viewing.  Through these activities, we presented the basic information about iGEM and synthetic biology to the youth and the public.</font>
+
-
</p>
+
<br><br>
<br><br>
 +
<div id="content1">
 +
<font face="impact" size="8" color="green">
 +
Polyphosphate Kinase<br><br>
 +
</font>
 +
<font face="century gothic" size="3">
 +
The enhanced biological phosphorous removal process is necessarily dependent on the ability of sludge microorganism (PAOs) to take up phosphate and to store it intracellularly in the form of polyP. For polyP formation to occur, phosphate must first be transported into the microbial cell and subsequently converted into ATP before incorporation in the polyP polymer. To improve the efficiency of the whole process, there are two steps we can work on:<br><br>
 +
(1) <b>Phosphate transport system</b>: increase phosphate uptake by engineering biological phosphate transport system.<br>
 +
(2) <b>PolyP synthesis</b>: engineer microbes to favour the formation of PolyP over the hydrolysis of PolyP, so that higher cell PolyP concentration and longer PolyP chain can be achieved.<br>
 +
</font>
-
<p><font face="Arial" size="5">Talk delivered to Sec School Students</font></p><br>
+
<br><br>
-
<div id="contentpicture">
+
<font face="impact" size="5" color="green">Phosphate transport system</font><br><br>
-
<img src="https://static.igem.org/mediawiki/2013/b/b8/B1a.jpg" height=auto width="280">
+
<font face="century gothic" size="3">
-
 
+
There are two major phosphate transport system in bacterial cells:<br><br>
-
</div>
+
A. The inorganic phosphate transport system (Pit) is constitutively expressed and has a relatively low specificity for phosphate. Pit transport neutral metal phosphates, each in symport with a proton.<br><br>
-
 
+
B. The phosphate-specific transport system (Pst) transport both H2PO4- and HPO42-, but not neutral metal phosphates. Unlike constitutively expressed Pit system, Pst system is phosphate-starvation inducible.<br><br>
-
<div id="contenttext">
+
More future research on microbial polyP metabolism in the EBPR process is needed to investigate how phosphate transport system activities affect polyP production. Clarification of the mechanism by which in-fluent phosphate is converted into ATP and used as substrate for polyP synthesis. Due to the much unknown details and limit of time, we choose to focus on the enhancement of PolyP synthesis efficiency inside bacteria.
-
<p>
+
-
<font face="Arial" size="2">
+
-
On Aug 10th, we gave a talk to 85 secondary school students from 12 schools who were the participants of a science video competition. The presentation consisted of an introduction about iGEM, synthetic biology and our project E. capsi. Since some of the audiences were from junior form, we used many cartoons and analogical examples to illustrate the basic concept such as BioBricks and synthetic biology.  Most students showed their interests in this project and some of them asked questions after the presentation.  We believe that giving presentation about iGEM to high school students, we, University students can inspire them to think deeply about how synthetic biology can change our lives.  
+
</font>
</font>
-
</p>  
+
<font face="impact" size="5" color="green"><br><br>PolyP synthesis</font><br><br>
 +
<img src="https://static.igem.org/mediawiki/2013/b/b9/Polyppppp.png" width="800" height=auto>
 +
<br>
 +
<font face="century gothic" size="3">
 +
Polyphosphate kinase 1 (PPK1) is the most extensively studied polyp-synthesizing enzyme and has been detected in a wide range of prokaryotes.  It catalyzes the transfer of the terminal phosphate of ATP to an active-site histidine residue, the initial step in the processive synthesis of a long PolyP chain. The reaction is reversible but favors synthesis.
 +
</font>
 +
<br>
 +
<img src="https://static.igem.org/mediawiki/parts/9/9e/Ppk1mol.png" width="250px" height=auto><br>
 +
<img src="https://static.igem.org/mediawiki/parts/c/c5/PPKequation.png" width=auto height="50px">
<br><br>
<br><br>
-
</div>
+
<font face="century gothic" size="3">
-
 
+
Although PPK’s involvement in the EBPR process is still unclear, we hypothesize over-expressing PPK can favour the PolyP synthesis and further increase the phosphate uptake. Hence we clone the ppk1 gene and put it under a controllable expression system.
 +
We notice that metabolic reactions are dynamic, maintaining a homeostasis inside the bacterial cells. PolyP synthesis by PPK1 may be counteracted by numerous “enemies” inside the bacteria.
 +
</font><br><br><br>
 +
<font face="impact" size="5" color="green">
 +
Enemies against PolyP synthesis<br><br>
 +
</font>
 +
<img src="https://static.igem.org/mediawiki/2013/2/2a/Degradepolypcartoon.png" width="800px" height=auto><br><br>
 +
<font face="century gothic" size="3">
 +
There are variety of hydrolases and phosphotransferases known to utilize polyP as a substrate in bacteria. Namely,<br><br>
 +
</font>
 +
<font face="century gothic" size="3" color="purple"><b>Exopolyphosphatase (PPX) (EC 3.6.1.11)</b><br>
 +
</font>
 +
<font face="century gothic" size="3">It is the major polyP-degrading enzyme in bacterial cells. This enzyme catalyzes the processive hydrolytic cleavage of Pi from the end of the polyP chain and the reaction may continue until only pyrophosphate (PPi) remains.</font>
 +
<br>
 +
<br>
 +
<img src=" https://static.igem.org/mediawiki/parts/c/c5/PPXequation.png" width=auto height="50px"><br><br>
 +
<br>
 +
<font face="century gothic" size="3" color="purple"><b>Polyphosphate Glucokinase (EC 2.7.1.63)</b><br></font>
 +
<font face="century gothic" size="3">It catalyzes an attack by glucose at the end of the polyP chain<br><br></font>
 +
<img src="https://static.igem.org/mediawiki/parts/0/0e/PiGluKinaseequation.png" width=auto height="50px"><br><br>
 +
<br>
 +
<font face="century gothic" size="3" color="purple"><b>AMP Phosphotransferase</b><br></font>
 +
<font face="century gothic" size="3">This enzyme catalyzes the attack at the end of the polyP chain by adenosine monophosphate (AMP) to produce ADP:<br><br></font>
 +
<img src="https://static.igem.org/mediawiki/parts/0/0e/PiGluKinaseequation.png" width=auto height="50px"><br><br>
 +
<font face="century gothic" size="3" color="purple"><b>ADP Phosphotransferase</b><br></font>
 +
<font face="century gothic" size="3">This enzyme is proposed to catalyse the polyphosphate-dependent phosphorylation of nucleoside diposphates such as ADP, the reverse activity of PPK:<br><br>
 +
<img src="https://static.igem.org/mediawiki/parts/e/e7/Reverseppkequation.png" width=auto height="50px"><br><br>
<br>
<br>
 +
Considering the vast number of polyP degradation pathways, we wonder, is there a way to isolate polyP synthesis from the surrounding polyP degrading enzymes? Just like the compartmentation in eukaryotes, can we also achieve compartmentation of metabolic reactions inside bacterial cells?<br>
 +
Hence, we come up with the idea of using</font> <font face="century gothic" size="3" color="green">Bacterial Micocompartment.<br></font>
-
<br><br>
 
-
<p><font face="Arial" size="5">Lab Tour for Biochemist Freshmen</font></p><br>
 
-
<br><br>
 
-
<div id="contentpicture">
 
-
<img src="https://static.igem.org/mediawiki/2013/7/75/Hkutalk.JPG" height=auto width="280">
 
-
</div>
 
-
<div id="contenttext">
 
-
<p>
 
-
<font face="Arial" size="2">
 
-
On Aug 24th, we had another seminar for newly admitted Biochemistry major undergraduate.  We also prepared some flyers about our project so they can have deeper understanding about iGEM competition.  The aim of the talk was to let students have better understanding about the applications and benefits of synthetic biology.  At the same time, we wanted to arouse their interests in this relatively new scientific field and encourage them to join iGEM competition in the future.  After the talk, the students were taken on a tour to visit the laboratory. 
 
-
</font>
 
-
</p>
 
-
</div>
 
-
<br><br>
 
 +
</font>
 +
<br><br>
</div>
</div>
</div>
</div>

Latest revision as of 03:06, 28 September 2013





Polyphosphate Kinase

The enhanced biological phosphorous removal process is necessarily dependent on the ability of sludge microorganism (PAOs) to take up phosphate and to store it intracellularly in the form of polyP. For polyP formation to occur, phosphate must first be transported into the microbial cell and subsequently converted into ATP before incorporation in the polyP polymer. To improve the efficiency of the whole process, there are two steps we can work on:

(1) Phosphate transport system: increase phosphate uptake by engineering biological phosphate transport system.
(2) PolyP synthesis: engineer microbes to favour the formation of PolyP over the hydrolysis of PolyP, so that higher cell PolyP concentration and longer PolyP chain can be achieved.


Phosphate transport system

There are two major phosphate transport system in bacterial cells:

A. The inorganic phosphate transport system (Pit) is constitutively expressed and has a relatively low specificity for phosphate. Pit transport neutral metal phosphates, each in symport with a proton.

B. The phosphate-specific transport system (Pst) transport both H2PO4- and HPO42-, but not neutral metal phosphates. Unlike constitutively expressed Pit system, Pst system is phosphate-starvation inducible.

More future research on microbial polyP metabolism in the EBPR process is needed to investigate how phosphate transport system activities affect polyP production. Clarification of the mechanism by which in-fluent phosphate is converted into ATP and used as substrate for polyP synthesis. Due to the much unknown details and limit of time, we choose to focus on the enhancement of PolyP synthesis efficiency inside bacteria.


PolyP synthesis



Polyphosphate kinase 1 (PPK1) is the most extensively studied polyp-synthesizing enzyme and has been detected in a wide range of prokaryotes. It catalyzes the transfer of the terminal phosphate of ATP to an active-site histidine residue, the initial step in the processive synthesis of a long PolyP chain. The reaction is reversible but favors synthesis.



Although PPK’s involvement in the EBPR process is still unclear, we hypothesize over-expressing PPK can favour the PolyP synthesis and further increase the phosphate uptake. Hence we clone the ppk1 gene and put it under a controllable expression system. We notice that metabolic reactions are dynamic, maintaining a homeostasis inside the bacterial cells. PolyP synthesis by PPK1 may be counteracted by numerous “enemies” inside the bacteria.


Enemies against PolyP synthesis



There are variety of hydrolases and phosphotransferases known to utilize polyP as a substrate in bacteria. Namely,

Exopolyphosphatase (PPX) (EC 3.6.1.11)
It is the major polyP-degrading enzyme in bacterial cells. This enzyme catalyzes the processive hydrolytic cleavage of Pi from the end of the polyP chain and the reaction may continue until only pyrophosphate (PPi) remains.




Polyphosphate Glucokinase (EC 2.7.1.63)
It catalyzes an attack by glucose at the end of the polyP chain




AMP Phosphotransferase
This enzyme catalyzes the attack at the end of the polyP chain by adenosine monophosphate (AMP) to produce ADP:



ADP Phosphotransferase
This enzyme is proposed to catalyse the polyphosphate-dependent phosphorylation of nucleoside diposphates such as ADP, the reverse activity of PPK:




Considering the vast number of polyP degradation pathways, we wonder, is there a way to isolate polyP synthesis from the surrounding polyP degrading enzymes? Just like the compartmentation in eukaryotes, can we also achieve compartmentation of metabolic reactions inside bacterial cells?
Hence, we come up with the idea of using
Bacterial Micocompartment.