Difference between revisions of "Team:NCKU Tainan/Design"

 
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                                         W3110 (K-12 laboratory strain) is reported to be resilient in a stressed environment.  
 
                                         W3110 (K-12 laboratory strain) is reported to be resilient in a stressed environment.  
 
                                         We expected that W3110 will grow well even if the sole carbon source is xylose.  
 
                                         We expected that W3110 will grow well even if the sole carbon source is xylose.  
                                         W3110(L5T7) (provided by Dr. Ng) is a constructed lab strain based on W3110.  
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                                         W3110 (L5T7) (provided by Dr. Ng) is a constructed lab strain based on W3110.  
 
                                         T7 polymerase was inserted into its genome.
 
                                         T7 polymerase was inserted into its genome.
 
                                     </p>
 
                                     </p>
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                                                 The optimized sequence was sent to IDT for gene synthesis.  
 
                                                 The optimized sequence was sent to IDT for gene synthesis.  
 
                                                 We PCR amplified the gene fragments and digest it with restriction enzymes HindIII and SpeI.  
 
                                                 We PCR amplified the gene fragments and digest it with restriction enzymes HindIII and SpeI.  
                                                 After digestion, we ligate the fragments into pSB3K3 plasmid with P<sub>LacI</sub>-rbs(B0034) located upstream of the fragment.  
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                                                 After digestion, we ligate the fragments into pSB3K3 plasmid with P<sub>LacI</sub>-rbs (B0034) located upstream of the fragment.  
 
                                                 The plasmid was then transformed into DH5 alpha.
 
                                                 The plasmid was then transformed into DH5 alpha.
 
                                             </p>
 
                                             </p>
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                                     <p class="pcontent">Ribulose-1,5-biphosphate carboxylase/oxygenase is one of the world’s most abundant enzyme.  
 
                                     <p class="pcontent">Ribulose-1,5-biphosphate carboxylase/oxygenase is one of the world’s most abundant enzyme.  
 
                                         It catalyzes the conversion of inorganic carbon into organic carbon.  
 
                                         It catalyzes the conversion of inorganic carbon into organic carbon.  
                                         In our designed pathway, the function of the Rubisco is to convert Ribulose-1,5-biphosphate (RuBP) from the upper pathway and carbon dioxide into 3-phosphoglycerate (3PGA).  
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                                         In our designed pathway, the function of the Rubisco is to convert ribulose-1,5-biphosphate (RuBP) from the upper pathway and carbon dioxide into 3-phosphoglycerate (3PGA).  
 
                                         3PGA will then be converted to pyruvate by the native metabolic system of <i>E. coli</i>.  
 
                                         3PGA will then be converted to pyruvate by the native metabolic system of <i>E. coli</i>.  
 
                                         After mining information from various publications,  
 
                                         After mining information from various publications,  
                                         we selected Rubisco from <i>Synechococcus elongatus</i> PCC7002, which is a well-studied cyanobacteria.  
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                                         we selected Rubisco from <i>Synechococcus elongatus</i> PCC 7002, which is a well-studied cyanobacteria.  
 
                                         Its genome is completely sequenced and it is often used as a model organism for gene manipulation.  
 
                                         Its genome is completely sequenced and it is often used as a model organism for gene manipulation.  
 
                                         Previous research has utilized <i>E. coli</i> as a host of random mutagenesis to enhance the activity of <i>Synechococcus</i> Rubisco.
 
                                         Previous research has utilized <i>E. coli</i> as a host of random mutagenesis to enhance the activity of <i>Synechococcus</i> Rubisco.
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                                     <p class="pcontent">Measurement of 3PGA or pyruvate concentration could not directly reflect the activity of Rubisco  
 
                                     <p class="pcontent">Measurement of 3PGA or pyruvate concentration could not directly reflect the activity of Rubisco  
 
                                         since both of them are important metabolites that will flow to downstream metabolic pathway.  
 
                                         since both of them are important metabolites that will flow to downstream metabolic pathway.  
                                         We then decided to determine its function by total solution test which we will mention below.
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                                         We then decided to determine its function by a total solution test which we will mention below.
 
                                     </p>
 
                                     </p>
 
                                 </div>
 
                                 </div>
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                                         Oxygen competes with CO<sub>2</sub> as a substrate for Rubisco, giving rise to photorespiration.  
 
                                         Oxygen competes with CO<sub>2</sub> as a substrate for Rubisco, giving rise to photorespiration.  
 
                                         To overcome this problem, some photosynthetic organisms have evolved their own carbon concentrating
 
                                         To overcome this problem, some photosynthetic organisms have evolved their own carbon concentrating
                                         mechanisms (CCM), which helps to maintain a sufficient amount of CO<sub>2</sub> around Rubisco.
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                                         mechanism (CCM), which helps to maintain a sufficient amount of CO<sub>2</sub> around Rubisco.
 
                                     </p>
 
                                     </p>
 
                                     <p class="pcontent">We are inspired by the carbon concentrating mechanisms (CCM) of cyanobacteria.  
 
                                     <p class="pcontent">We are inspired by the carbon concentrating mechanisms (CCM) of cyanobacteria.  
 
                                         In cyanobacteria, Rubisco and carbonic anhydrase (CA) is encapsulated in a microcompartment, the carboxysome.  
 
                                         In cyanobacteria, Rubisco and carbonic anhydrase (CA) is encapsulated in a microcompartment, the carboxysome.  
 
                                         Carbonic anhydrase, also known as carbonate dehydratase, is involved in the interconversion between CO<sub>2</sub> and HCO<sub>3</sub><sup>-</sup>. This enzyme can be found in most organisms, including <i>E. coli</i> but the difference is its catalyzing rate in hydration and dehydration of CO2. Therefore,  
 
                                         Carbonic anhydrase, also known as carbonate dehydratase, is involved in the interconversion between CO<sub>2</sub> and HCO<sub>3</sub><sup>-</sup>. This enzyme can be found in most organisms, including <i>E. coli</i> but the difference is its catalyzing rate in hydration and dehydration of CO2. Therefore,  
                                         we will incorporate into our system the carbonic anhydrase gene from <i>Synechococcus elongatus</i> PCC7002.
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                                         we will incorporate into our system the carbonic anhydrase gene from <i>Synechococcus elongatus</i> PCC 7002.
 
                                     </p>
 
                                     </p>
 
                                     <img class="gif" src="https://static.igem.org/mediawiki/2018/3/34/T--NCKU_Tainan--design_CA.gif" alt="Rubisco">
 
                                     <img class="gif" src="https://static.igem.org/mediawiki/2018/3/34/T--NCKU_Tainan--design_CA.gif" alt="Rubisco">
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                                             <p class="pcontent">We first codon optimized the sequence and insert it into the empty pSB1C3 plasmid with HindIII and  
 
                                             <p class="pcontent">We first codon optimized the sequence and insert it into the empty pSB1C3 plasmid with HindIII and  
 
                                                 SpeI just as mentioned above. In our optimized sequence, we have already designed a P<sub>T7</sub> promoter  
 
                                                 SpeI just as mentioned above. In our optimized sequence, we have already designed a P<sub>T7</sub> promoter  
                                                 in front of CA, so we can directly ligate it into the plasmid.  
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                                                 in front of <i>ca</i>, so we can directly ligate it into the plasmid.  
 
                                                 The constructed basic part is then linked with other basic parts to complete our construction.
 
                                                 The constructed basic part is then linked with other basic parts to complete our construction.
 
                                             </p>
 
                                             </p>
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                                     <p class="pcontent">To measure the enzyme activity of CA, we compare the conversion rate of carbon dioxide to  
 
                                     <p class="pcontent">To measure the enzyme activity of CA, we compare the conversion rate of carbon dioxide to  
 
                                         bicarbonate ion. After saturated CO<sub>2</sub> solution is prepared, we add fixed amount of bacteria broth that  
 
                                         bicarbonate ion. After saturated CO<sub>2</sub> solution is prepared, we add fixed amount of bacteria broth that  
                                         contains CA construction into the solution. We then measure the time interval of the decrease pH value from 8.3 to 6.3.  
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                                         contains CA construction into the solution. We then measure the time taken for the pH value to decrease from 8.3 to 6.3.  
 
                                         We compare the measured time interval with the time interval that enzyme was not added to determine the enzyme activity of CA.
 
                                         We compare the measured time interval with the time interval that enzyme was not added to determine the enzyme activity of CA.
 
                                     </p>
 
                                     </p>
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                                         Previously, every basic part was the backbone conserved in the backbone of pSB1C3.  
 
                                         Previously, every basic part was the backbone conserved in the backbone of pSB1C3.  
 
                                         We then link the construction together and  
 
                                         We then link the construction together and  
                                         even change the backbone of some composite parts to pSB3K3 for lower protein expression.
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                                         even change the backbone of some composite parts to pSB3K3 for a lower protein expression.
 
                                     </p>
 
                                     </p>
 
                                     <h5 class="question">Rubisco whole protein in pSB1C3</h5>
 
                                     <h5 class="question">Rubisco whole protein in pSB1C3</h5>
                                     <p class="pcontent">We link each basic parts with biobrick standard method.  
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                                     <p class="pcontent">We link each basic part together with biobrick standard method.  
 
                                         We link P<sub>T7</sub>-<i>rbcL</i> and P<sub>T7</sub>-<i>rbcX</i>-<i>rbcS</i> together.  
 
                                         We link P<sub>T7</sub>-<i>rbcL</i> and P<sub>T7</sub>-<i>rbcX</i>-<i>rbcS</i> together.  
                                         The former, the insert, was digested with EcoRI and SpeI and the later, the backbone,  
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                                         The former, the insert, was digested with EcoRI and SpeI and the latter, the backbone,  
 
                                         is digested with EcoRI and XbaI.  
 
                                         is digested with EcoRI and XbaI.  
 
                                         We ligate the backbone with the insert to complete this composite part.
 
                                         We ligate the backbone with the insert to complete this composite part.
 
                                     </p>
 
                                     </p>
 
                                     <h5 class="question"><i>prk</i> gene into pSB3K3</h5>
 
                                     <h5 class="question"><i>prk</i> gene into pSB3K3</h5>
                                     <p class="pcontent">PRK catalyzes the reaction of turning Ru5P into RuBP.  
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                                     <p class="pcontent">PRK catalyzes the reaction of converting Ru5P into RuBP.  
 
                                         Not native to the host, RuBP is, nonetheless, toxic to <i>E. coli</i>.  
 
                                         Not native to the host, RuBP is, nonetheless, toxic to <i>E. coli</i>.  
                                         We hope that expression of PRK to be lower in the host so we change the backbone of it into pSB3K3.  
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                                         We hope that the expression of PRK could be lower in the host so we change the backbone of it into pSB3K3.  
 
                                         We selected J04450 from the distributed kit that under the backbone of pSB3K3,  
 
                                         We selected J04450 from the distributed kit that under the backbone of pSB3K3,  
 
                                         which will express red color after the formation of the colony. We digest both backbone and insert with EcoRI and PstI and ligate both fragments.  
 
                                         which will express red color after the formation of the colony. We digest both backbone and insert with EcoRI and PstI and ligate both fragments.  
 
                                         We can then select the colony that does not present red color to prove that the ligation was conducted successfully.
 
                                         We can then select the colony that does not present red color to prove that the ligation was conducted successfully.
 
                                     </p>
 
                                     </p>
                                     <h5 class="question">Link <i>prk</i> with CA into pSB3K3</h5>
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                                     <h5 class="question">Link <i>prk</i> with <i>ccaA</i> into pSB3K3</h5>
                                     <p class="pcontent">We also constructed the composite part that contains both CA and <i>prk</i>.  
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                                     <p class="pcontent">We also constructed the composite part that contains both <i>ccaA</i> and <i>prk</i>.  
 
                                         We construct it using the method mentioned in <i>rbc</i> whole construction.  
 
                                         We construct it using the method mentioned in <i>rbc</i> whole construction.  
 
                                         We cloned the fragments into pSB3K3 for lower expression of PRK.
 
                                         We cloned the fragments into pSB3K3 for lower expression of PRK.
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                                     <h5 class="question">Transformation</h5>
 
                                     <h5 class="question">Transformation</h5>
 
                                     <p class="pcontent">After the construction of various composite parts,  
 
                                     <p class="pcontent">After the construction of various composite parts,  
                                         we co-transform them into three <i>E. coli</i> strains: BL21(DE3), W3110, and W3110(L5T7).  
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                                         we co-transform them into three <i>E. coli</i> strains: BL21 (DE3), W3110, and W3110 (L5T7).  
                                         Since BL21(DE3) and W3110(L5T7) contains T7 polymerase,  
+
                                         Since BL21 (DE3) and W3110 (L5T7) contains T7 polymerase,  
 
                                         we co-transformed composite parts that contain T7 promoter into these strains.  
 
                                         we co-transformed composite parts that contain T7 promoter into these strains.  
 
                                         We co-transform plasmid that only contains LacI promoter into W3110.
 
                                         We co-transform plasmid that only contains LacI promoter into W3110.
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                                     <h5 class="question">How to prove our design?</h5>
 
                                     <h5 class="question">How to prove our design?</h5>
 
                                     <p class="pcontent">We designed a total solution test to verify the function of our whole construction.  
 
                                     <p class="pcontent">We designed a total solution test to verify the function of our whole construction.  
                                         We incubate the constructed strains in modified M9 medium that contains 0.4% xylose as its sole carbon source.  
+
                                         We incubate the constructed strains in modified M9 medium that contains 4 (g/l) xylose as its sole carbon source.  
 
                                         The construction is designed to consume xylose as energy source and as a material for Calvin-Benson cycle.  
 
                                         The construction is designed to consume xylose as energy source and as a material for Calvin-Benson cycle.  
 
                                         We then measure the optical intensity (O.D. 600) to characterize the cell growth. At a fixed time interval,  
 
                                         We then measure the optical intensity (O.D. 600) to characterize the cell growth. At a fixed time interval,  
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                                                 We then exchanged the promoter with the previously constructed plasmid that contains P<sub>T7</sub> and GFP or sfGFP.  
 
                                                 We then exchanged the promoter with the previously constructed plasmid that contains P<sub>T7</sub> and GFP or sfGFP.  
 
                                                 We initially transformed the constructed plasmid into DH5 alpha for colony screening.  
 
                                                 We initially transformed the constructed plasmid into DH5 alpha for colony screening.  
                                                 We then transformed the plasmid into BL21(DE3) to test its function.
+
                                                 We then transformed the plasmid into BL21 (DE3) to test its function.
 
                                                 We also design another biobrick that contains riboJ (a signal amplify fragment)  
 
                                                 We also design another biobrick that contains riboJ (a signal amplify fragment)  
 
                                                 at the downstream of P<sub>gadA</sub> to get the signal more clearly and enhance the specificity.
 
                                                 at the downstream of P<sub>gadA</sub> to get the signal more clearly and enhance the specificity.

Latest revision as of 15:18, 3 November 2018

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