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<div class="righttitle"> | <div class="righttitle"> | ||
− | <h6 class="subtitle"> | + | <h6 class="subtitle">Bring Solutions, Not Problems</h6> |
</div> | </div> | ||
<div class="navbar-example"> | <div class="navbar-example"> | ||
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Plant, algae, and cyanobacteria utilize light as energy source for Calvin-Benson cycle. | Plant, algae, and cyanobacteria utilize light as energy source for Calvin-Benson cycle. | ||
Taking the advantage of the pentose phosphate pathway, a native metabolic pathway of <i>E. coli</i>, | Taking the advantage of the pentose phosphate pathway, a native metabolic pathway of <i>E. coli</i>, | ||
− | only two additional enzymes will be needed to reconstruct the pathway in <i>E. coli</i> -- PRK and | + | only two additional enzymes will be needed to reconstruct the pathway in <i>E. coli</i> -- PRK and Rubisco, |
which we will describe more in detail. | which we will describe more in detail. | ||
The primary product of the pathway is pyruvate, which can be utilized to produce various valuable products. | The primary product of the pathway is pyruvate, which can be utilized to produce various valuable products. | ||
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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. | + | 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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<div class="card card-body"> | <div class="card card-body"> | ||
<p class="pcontent">Steps involved in expressing PRK in <i>E. coli.</i> | <p class="pcontent">Steps involved in expressing PRK in <i>E. coli.</i> | ||
− | We initially confirm the gene sequence of <i>Synechococcus elongtus</i> | + | We initially confirm the gene sequence of <i>Synechococcus elongtus</i> <i>prk</i> from NCBI gene database. |
We then codon optimized the sequence so <i>E. coli</i> can express the protein properly. | We then codon optimized the sequence so <i>E. coli</i> can express the protein properly. | ||
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. | + | 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 | + | 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 | + | 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> | + | Previous research has utilized <i>E. coli</i> as a host of random mutagenesis to enhance the activity of <i>Synechococcus</i> Rubisco. |
</p> | </p> | ||
<img class="gif" src="https://static.igem.org/mediawiki/2018/8/85/T--NCKU_Tainan--design_Rubisco.gif" alt="Rubisco"> | <img class="gif" src="https://static.igem.org/mediawiki/2018/8/85/T--NCKU_Tainan--design_Rubisco.gif" alt="Rubisco"> | ||
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<div class="collapse multi-collapse" id="RuBisCO_how_to_construct"> | <div class="collapse multi-collapse" id="RuBisCO_how_to_construct"> | ||
<div class="card card-body"> | <div class="card card-body"> | ||
− | <p class="pcontent">Akin to the construction of | + | <p class="pcontent">Akin to the construction of <i>prk</i>, we codon optimized the sequence of three <i>rbc</i> subunit and |
clone it into pSB1C3 plasmid with HindIII and SpeI. | clone it into pSB1C3 plasmid with HindIII and SpeI. | ||
− | The sequence and the size of | + | The sequence and the size of <i>rbcL</i> is much larger than other subunit, |
− | so we separated rbcL from rbcX and rbcS subunits. | + | so we separated <i>rbcL</i> from <i>rbcX</i> and <i>rbcS</i> subunits. <i>rbcX</i> and <i>rbcS</i> is separated by a <i>rbs</i> (B0034) for the convenience of construction. |
− | We attached two different promoters upstream of the | + | We attached two different promoters upstream of the <i>rbc</i>. They are P<sub>LacI</sub> and P<sub>T7</sub> promoter. |
Since we would like to increase the expression of this protein in the metabolic pathway, | Since we would like to increase the expression of this protein in the metabolic pathway, | ||
we would like to test various promoter combination to find out the most efficient combination for our pathway. | we would like to test various promoter combination to find out the most efficient combination for our pathway. | ||
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<img class="bigimg" src="https://static.igem.org/mediawiki/2018/a/ad/T--NCKU_Tainan--design_RBC_construction.png" alt="RBC construction picture"> | <img class="bigimg" src="https://static.igem.org/mediawiki/2018/a/ad/T--NCKU_Tainan--design_RBC_construction.png" alt="RBC construction picture"> | ||
<h5 class="question">How do we test its function?</h5> | <h5 class="question">How do we test its function?</h5> | ||
− | <p class="pcontent">Measurement of 3PGA or pyruvate concentration could not directly reflect the activity of | + | <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. | + | 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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<h3>CA</h3> | <h3>CA</h3> | ||
<h5 class="question">What is its function?</h5> | <h5 class="question">What is its function?</h5> | ||
− | <p class="pcontent"> | + | <p class="pcontent">Rubisco is the rate-limiting enzyme in carbon fixation. |
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 | ||
− | + | 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> | + | 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 | + | 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 | + | 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. | + | 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 | + | <p class="pcontent">We link each basic part together with biobrick standard method. |
− | We link P<sub>T7</sub>-rbcL and P<sub>T7</sub>-rbcX-rbcS 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 | + | 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"> | + | <h5 class="question"><i>prk</i> gene into pSB3K3</h5> |
− | <p class="pcontent">PRK catalyzes the reaction of | + | <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 | + | 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 | + | <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 | + | <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 | + | 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. | ||
</p> | </p> | ||
<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). | + | 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 | + | 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. | ||
</p> | </p> | ||
<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 | + | 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