Difference between revisions of "Team:UChile-OpenBio/Notebook"

Line 40: Line 40:
 
             <!-- SI EXPORT END -->
 
             <!-- SI EXPORT END -->
 
                
 
                
−
                 <span style="background:#66FF66" class="titulo_seccion">Notebook</span>
+
 
 +
<section id="weeks">
 +
                 <span style="background:#33CC33" class="titulo_seccion">Notebook</span>
 
                 <article>
 
                 <article>
 
                     <div class="division">
 
                     <div class="division">
 
                         <div class="half">
 
                         <div class="half">
 
<!-------COLUMNA 1-->
 
<!-------COLUMNA 1-->
−
                         <h2><span style="color:#66FF66">Week 1: The beginning</span></h2>
+
                         <h2><span style="color:#33CC33">Week 1: The beginning</span></h2>
 
                        
 
                        
 
<p> We formed as an iGEM group in 2014, when we started to have recurrent meeting for creating new ideas of possible projects of synthetic biology, including some projects in the Centre of Bioengineering and Biotechnology. Our first meeting was several brainstorming considering two main objectives: to generate new ideas of projects and assess the feasibility in terms of experimental design. </p>
 
<p> We formed as an iGEM group in 2014, when we started to have recurrent meeting for creating new ideas of possible projects of synthetic biology, including some projects in the Centre of Bioengineering and Biotechnology. Our first meeting was several brainstorming considering two main objectives: to generate new ideas of projects and assess the feasibility in terms of experimental design. </p>
 
   
 
   
−
<p>The generated ideas was related to solve problem of routine laboratory methodologies, sustentabilities issues, and other problems of molecular biology. After several brainstorming activities we gather several of project that needed to be processed and assessed its feasibility. One of the meetings of the recently formed iGEM group, in that time called just “OpenBio”, we met the group Fab-LAB and they were interested in a new form of producing a raw material used for 3D printing, Polylactic Acid (PLA). Until that we saw different ways of how to produce polymers of different kind in a biological system, comprehending the metabolic pathways known and searching for new genetic sources. Then, the project of PLA production in a biological system using synthetic biology tools is formed.</p>
+
<p>The generated ideas was related to solve problem of routine laboratory methodologies, sustentabilities issues, and other problems of molecular biology. After several brainstorming activities we gather several of project that needed to be processed and assessed its feasibility. One of the meetings of the recently formed iGEM group, in that time called just “OpenBio”, we met the group Fab-LAB and they were interested in a new form of producing a raw material used for 3D printing, Polylactic Acid (PLA). Until that we saw different ways of how to produce polymers of different kind in a biological system, comprehending the metabolic pathways known and searching for new genetic sources. Then, the project of PLA production in a biological system using synthetic biology tools is formed.</p><br>
  
 +
                        </div>
 +
                        <div class="half last">
 +
                         
 +
<!-------COLUMNA 2-->
 +
<h2><span style="color:#33CC33">Week 2: Preparation of the design and complementary activities</span></h2>
  
−
                      <h2><span style="color:#66FF66">Week 2: Preparation of the design and complementary activities</span></h2>
+
<p>After the formation of the PLA project was elected by unanimity, we started to work the design of the project and the molecular biology needed to achieve a biological system controlled by a regulatory network that accomplished the principles of synthetic biology. Then, the first ideas of a regulatory network has been created using the microorganism <em>E. coli</em> as a host. PLA is actually produced by chemical synthesis using an old system called O’Ring polymerization, so then a biological system should have comparable yields and low cost of purification process. Then the first conception was to enable a biological system that could handle a large production of recombinant proteins derived of a synthetic pathway, and that could be achieved using a controlled system based in the kinetic growth of the microorganism. Thus, a quorum sensing based expression system could handle the metabolic burden involved in a new pathway, and the production of PLA could be sustainable in time. The second concept is to improve the extraction processes of PLA after fermentations. Lately, biological production of PLA consider harvesting cell pellet and then destroy cell membrane to extract intracellular PLA. The significant costs associated to this process could be improved implementing a PLA export system that could be similar to a PHA phasins export systems reported. So the goal of the project y to design all of these genetic circuits properly regulated by the cell growth and the other physiological conditions.</p><br>
  
−
<p>After the formation of the PLA project was elected by unanimity, we started to work the design of the project and the molecular biology needed to achieve a biological system controlled by a regulatory network that accomplished the principles of synthetic biology. Then, the first ideas of a regulatory network has been created using the microorganism <em>E. coli</em> as a host. PLA is actually produced by chemical synthesis using an old system called O’Ring polymerization, so then a biological system should have comparable yields and low cost of purification process. Then the first conception was to enable a biological system that could handle a large production of recombinant proteins derived of a synthetic pathway, and that could be achieved using a controlled system based in the kinetic growth of the microorganism. Thus, a quorum sensing based expression system could handle the metabolic burden involved in a new pathway, and the production of PLA could be sustainable in time. The second concept is to improve the extraction processes of PLA after fermentations. Lately, biological production of PLA consider harvesting cell pellet and then destroy cell membrane to extract intracellular PLA. The significant costs associated to this process could be improved implementing a PLA export system that could be similar to a PHA phasins export systems reported. So the goal of the project y to design all of these genetic circuits properly regulated by the cell growth and the other physiological conditions.</p>
+
                        </div>
 +
                    </div>
 +
                </article>
  
  
  
 +
                <article>
 +
                    <div class="division">
 +
                        <div class="half">
 +
<!-------COLUMNA 1-->
 +
                        <h2><span style="color:#33CC33">Week 3: The design </span></h2>
 +
                     
 +
<p> The design of the project is divided in three stages. First the design of the genetic circuits from different biological sources. We first define the synthesis of PLA and lactic acid with the currently conventional gene codifying enzymes and mutated ones. The main enzymes are poly-hydroxyalcanoate synthase (PhaC) PLA polymerization and propionyl-CoA-Transferase (P-Co-A-T) for lactyl-CoA substrate production. We recently we decided to synthesize and assembly the whole metabolic pathway of PLA production regulated by quorum sensing and PLA exportation using gBlocks assembly from IDT technologies. </p>
 +
 +
<p>The generated ideas was related to solve problem of routine laboratory methodologies, sustentabilities issues, and other problems of molecular biology. After several brainstorming activities we gather several of project that needed to be processed and assessed its feasibility. One of the meetings of the recently formed iGEM group, in that time called just “OpenBio”, we met the group Fab-LAB and they were interested in a new form of producing a raw material used for 3D printing, Polylactic Acid (PLA). Until that we saw different ways of how to produce polymers of different kind in a biological system, comprehending the metabolic pathways known and searching for new genetic sources. Then, the project of PLA production in a biological system using synthetic biology tools is formed.</p><br>
  
 +
                        </div>
 +
                        <div class="half last">
 +
                         
 +
<!-------COLUMNA 2-->
 +
                        <h2><span style="color:#33CC33">Week 4: Design of gblock for ordering synthesis</span></h2>
  
 +
<p>After the formation of the PLA project was elected by unanimity, we started to work the design of the project and the molecular biology needed to achieve a biological system controlled by a regulatory network that accomplished the principles of synthetic biology. Then, the first ideas of a regulatory network has been created using the microorganism <em>E. coli</em> as a host. PLA is actually produced by chemical synthesis using an old system called O’Ring polymerization, so then a biological system should have comparable yields and low cost of purification process. Then the first conception was to enable a biological system that could handle a large production of recombinant proteins derived of a synthetic pathway, and that could be achieved using a controlled system based in the kinetic growth of the microorganism. Thus, a quorum sensing based expression system could handle the metabolic burden involved in a new pathway, and the production of PLA could be sustainable in time. The second concept is to improve the extraction processes of PLA after fermentations. Lately, biological production of PLA consider harvesting cell pellet and then destroy cell membrane to extract intracellular PLA. The significant costs associated to this process could be improved implementing a PLA export system that could be similar to a PHA phasins export systems reported. So the goal of the project y to design all of these genetic circuits properly regulated by the cell growth and the other physiological conditions.</p><br>
  
 +
                        </div>
 +
                    </div>
 +
                </article>
  
  
  
  
 +
 +
                <article>
 +
                    <div class="division">
 +
                        <div class="half">
 +
<!-------COLUMNA 1-->
 +
                        <h2><span style="color:#33CC33">Week 5: First experiments </span></h2>
 +
                     
 +
<p> The Gibson assembly g blocks has arrived!, So the first experiments will be performed.  The order was read carefully and the incubation of gBlocks was done by the manufacturer instructions. All the gBlocks consist in the parts required for the genetic circuit and the some of them must be assembled by Gibson assembly, especially the gene codifying enzymes of the metabolic pathway.  Unfortunately we had problems with gBlocks, because apparently the concentration was much lower than the specified. This issue produce a delay in our program of activities, when we decided to re-amplify them designing and ordering new primers. </p>
 +
 +
<p>We tried to calculate de concentration of all the gBlocks to proceed the assembly, unfortunately y we would no be able to complete, because of low concentration of templates. The first Gibson assembly of Templates was LDH gblocks, with low concentration we fairly could able to generate the desired product (1237 bp).  Other assemblies like LuxI failed in the assembly attempt.</p><br>
  
 
                         </div>
 
                         </div>
Line 70: Line 107:
 
                            
 
                            
 
<!-------COLUMNA 2-->
 
<!-------COLUMNA 2-->
 +
                        <h2><span style="color:#33CC33">Week 4: Design of gblock for ordering synthesis</span></h2>
 +
 +
<p>After the formation of the PLA project was elected by unanimity, we started to work the design of the project and the molecular biology needed to achieve a biological system controlled by a regulatory network that accomplished the principles of synthetic biology. Then, the first ideas of a regulatory network has been created using the microorganism <em>E. coli</em> as a host. PLA is actually produced by chemical synthesis using an old system called O’Ring polymerization, so then a biological system should have comparable yields and low cost of purification process. Then the first conception was to enable a biological system that could handle a large production of recombinant proteins derived of a synthetic pathway, and that could be achieved using a controlled system based in the kinetic growth of the microorganism. Thus, a quorum sensing based expression system could handle the metabolic burden involved in a new pathway, and the production of PLA could be sustainable in time. The second concept is to improve the extraction processes of PLA after fermentations. Lately, biological production of PLA consider harvesting cell pellet and then destroy cell membrane to extract intracellular PLA. The significant costs associated to this process could be improved implementing a PLA export system that could be similar to a PHA phasins export systems reported. So the goal of the project y to design all of these genetic circuits properly regulated by the cell growth and the other physiological conditions.</p><br>
 +
 
                         </div>
 
                         </div>
 
                     </div>
 
                     </div>
 
                 </article>
 
                 </article>
 +
 +
 +
 +
 +
</section>
 +
 +
  
  

Revision as of 22:13, 18 September 2015


TODO supply a title

Notebook

Week 1: The beginning

We formed as an iGEM group in 2014, when we started to have recurrent meeting for creating new ideas of possible projects of synthetic biology, including some projects in the Centre of Bioengineering and Biotechnology. Our first meeting was several brainstorming considering two main objectives: to generate new ideas of projects and assess the feasibility in terms of experimental design.

The generated ideas was related to solve problem of routine laboratory methodologies, sustentabilities issues, and other problems of molecular biology. After several brainstorming activities we gather several of project that needed to be processed and assessed its feasibility. One of the meetings of the recently formed iGEM group, in that time called just “OpenBio”, we met the group Fab-LAB and they were interested in a new form of producing a raw material used for 3D printing, Polylactic Acid (PLA). Until that we saw different ways of how to produce polymers of different kind in a biological system, comprehending the metabolic pathways known and searching for new genetic sources. Then, the project of PLA production in a biological system using synthetic biology tools is formed.


Week 2: Preparation of the design and complementary activities

After the formation of the PLA project was elected by unanimity, we started to work the design of the project and the molecular biology needed to achieve a biological system controlled by a regulatory network that accomplished the principles of synthetic biology. Then, the first ideas of a regulatory network has been created using the microorganism E. coli as a host. PLA is actually produced by chemical synthesis using an old system called O’Ring polymerization, so then a biological system should have comparable yields and low cost of purification process. Then the first conception was to enable a biological system that could handle a large production of recombinant proteins derived of a synthetic pathway, and that could be achieved using a controlled system based in the kinetic growth of the microorganism. Thus, a quorum sensing based expression system could handle the metabolic burden involved in a new pathway, and the production of PLA could be sustainable in time. The second concept is to improve the extraction processes of PLA after fermentations. Lately, biological production of PLA consider harvesting cell pellet and then destroy cell membrane to extract intracellular PLA. The significant costs associated to this process could be improved implementing a PLA export system that could be similar to a PHA phasins export systems reported. So the goal of the project y to design all of these genetic circuits properly regulated by the cell growth and the other physiological conditions.


Week 3: The design

The design of the project is divided in three stages. First the design of the genetic circuits from different biological sources. We first define the synthesis of PLA and lactic acid with the currently conventional gene codifying enzymes and mutated ones. The main enzymes are poly-hydroxyalcanoate synthase (PhaC) PLA polymerization and propionyl-CoA-Transferase (P-Co-A-T) for lactyl-CoA substrate production. We recently we decided to synthesize and assembly the whole metabolic pathway of PLA production regulated by quorum sensing and PLA exportation using gBlocks assembly from IDT technologies.

The generated ideas was related to solve problem of routine laboratory methodologies, sustentabilities issues, and other problems of molecular biology. After several brainstorming activities we gather several of project that needed to be processed and assessed its feasibility. One of the meetings of the recently formed iGEM group, in that time called just “OpenBio”, we met the group Fab-LAB and they were interested in a new form of producing a raw material used for 3D printing, Polylactic Acid (PLA). Until that we saw different ways of how to produce polymers of different kind in a biological system, comprehending the metabolic pathways known and searching for new genetic sources. Then, the project of PLA production in a biological system using synthetic biology tools is formed.


Week 4: Design of gblock for ordering synthesis

After the formation of the PLA project was elected by unanimity, we started to work the design of the project and the molecular biology needed to achieve a biological system controlled by a regulatory network that accomplished the principles of synthetic biology. Then, the first ideas of a regulatory network has been created using the microorganism E. coli as a host. PLA is actually produced by chemical synthesis using an old system called O’Ring polymerization, so then a biological system should have comparable yields and low cost of purification process. Then the first conception was to enable a biological system that could handle a large production of recombinant proteins derived of a synthetic pathway, and that could be achieved using a controlled system based in the kinetic growth of the microorganism. Thus, a quorum sensing based expression system could handle the metabolic burden involved in a new pathway, and the production of PLA could be sustainable in time. The second concept is to improve the extraction processes of PLA after fermentations. Lately, biological production of PLA consider harvesting cell pellet and then destroy cell membrane to extract intracellular PLA. The significant costs associated to this process could be improved implementing a PLA export system that could be similar to a PHA phasins export systems reported. So the goal of the project y to design all of these genetic circuits properly regulated by the cell growth and the other physiological conditions.


Week 5: First experiments

The Gibson assembly g blocks has arrived!, So the first experiments will be performed. The order was read carefully and the incubation of gBlocks was done by the manufacturer instructions. All the gBlocks consist in the parts required for the genetic circuit and the some of them must be assembled by Gibson assembly, especially the gene codifying enzymes of the metabolic pathway. Unfortunately we had problems with gBlocks, because apparently the concentration was much lower than the specified. This issue produce a delay in our program of activities, when we decided to re-amplify them designing and ordering new primers.

We tried to calculate de concentration of all the gBlocks to proceed the assembly, unfortunately y we would no be able to complete, because of low concentration of templates. The first Gibson assembly of Templates was LDH gblocks, with low concentration we fairly could able to generate the desired product (1237 bp). Other assemblies like LuxI failed in the assembly attempt.


Week 4: Design of gblock for ordering synthesis

After the formation of the PLA project was elected by unanimity, we started to work the design of the project and the molecular biology needed to achieve a biological system controlled by a regulatory network that accomplished the principles of synthetic biology. Then, the first ideas of a regulatory network has been created using the microorganism E. coli as a host. PLA is actually produced by chemical synthesis using an old system called O’Ring polymerization, so then a biological system should have comparable yields and low cost of purification process. Then the first conception was to enable a biological system that could handle a large production of recombinant proteins derived of a synthetic pathway, and that could be achieved using a controlled system based in the kinetic growth of the microorganism. Thus, a quorum sensing based expression system could handle the metabolic burden involved in a new pathway, and the production of PLA could be sustainable in time. The second concept is to improve the extraction processes of PLA after fermentations. Lately, biological production of PLA consider harvesting cell pellet and then destroy cell membrane to extract intracellular PLA. The significant costs associated to this process could be improved implementing a PLA export system that could be similar to a PHA phasins export systems reported. So the goal of the project y to design all of these genetic circuits properly regulated by the cell growth and the other physiological conditions.


Our Sponsors

UChile Sponsors