(Blanked the page) |
|||
(71 intermediate revisions by 5 users not shown) | |||
Line 1: | Line 1: | ||
− | + | {{NCKU_Tainan/header}} {{NCKU_Tainan/navbar}} {{NCKU_Tainan/style}} | |
+ | <html> | ||
+ | <head> | ||
+ | <link rel="stylesheet" href="https://2018.igem.org/Template:NCKU_Tainan/css/design?action=raw&ctype=text/css"> | ||
+ | </head> | ||
+ | <body data-spy="scroll" data-target=".navbar-example"> | ||
+ | <div class="container content"> | ||
+ | <div class="headstyle"> | ||
+ | <h1 class="head">Design</h1> | ||
+ | </div> | ||
+ | <div class="righttitle"> | ||
+ | <h6 class="subtitle">Bring Solutions, Not Problems</h6> | ||
+ | </div> | ||
+ | <div class="navbar-example"> | ||
+ | <div class="row"> | ||
+ | <div class="col-2 side"> | ||
+ | <div id="sidelist" class="list-group"> | ||
+ | <a class="list-group-item list-group-item-action" href="#Overview">Overview</a> | ||
+ | <a class="list-group-item list-group-item-action" href="#Chassis_Organism">Chassis Organism</a> | ||
+ | <a class="list-group-item list-group-item-action" href="#Prk">Prk</a> | ||
+ | <a class="list-group-item list-group-item-action" href="#Rubisco">Rubisco</a> | ||
+ | <a class="list-group-item list-group-item-action" href="#CA">CA</a> | ||
+ | <a class="list-group-item list-group-item-action" href="#dual_plasmid_system">Dual plasmid system</a> | ||
+ | <a class="list-group-item list-group-item-action" href="#pH_sensing_system">pH sensing system</a> | ||
+ | <a class="list-group-item list-group-item-action" href="#Reference">References</a> | ||
+ | <a class="list-group-item list-group-item-action" href="#"><i class="fa fa-arrow-up fa-1x" aria-hidden="true"></i></a> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="col-10"> | ||
+ | <div data-spy="scroll" data-target="#sidelist" data-offset="0" class="scrollspy-example"> | ||
+ | <div class="container"> | ||
+ | <div id="Overview"> | ||
+ | <h3>Overview of Designed Pathway</h3> | ||
+ | <img class="gif" src="https://static.igem.org/mediawiki/2018/2/2c/T--NCKU_Tainan--wetdesign.gif" alt="Total pathway"> | ||
+ | <p class="pcontent">Calvin-Benson cycle is one of the most important pathways for inorganic carbon to be converted into organic carbon in the carbon 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>, | ||
+ | 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. | ||
+ | The primary product of the pathway is pyruvate, which can be utilized to produce various valuable products. | ||
+ | </p> | ||
+ | <img class="gif" src="https://static.igem.org/mediawiki/2018/3/30/T--NCKU_Tainan--design_totalpathway.gif" alt="Total pathway"> | ||
+ | </div> | ||
+ | <div id="Chassis_Organism"> | ||
+ | <h3>Chassis Organism</h3> | ||
+ | <p class="pcontent">We would like to test our pathway in various <i>E. coli</i> strains to see the functionality of our construction. | ||
+ | We selected three different strains: BL21 (DE3), W3110, W3110 (L5T7). | ||
+ | BL21 (DE3) is a common expression strain that is widely used to express recombinant proteins using T7 polymerase. | ||
+ | We expected that the high production of protein may change the entire native metabolic pathway. | ||
+ | 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. | ||
+ | W3110 (L5T7) (provided by Dr. Ng) is a constructed lab strain based on W3110. | ||
+ | T7 polymerase was inserted into its genome. | ||
+ | </p> | ||
+ | </div> | ||
+ | <div id="Prk"> | ||
+ | <h3>PRK</h3> | ||
+ | <h5 class="question">What is its function?</h5> | ||
+ | <p class="pcontent">We first introduced phosphoribulokinase (PRK), an enzyme from cyanobacterial Calvin cycle, | ||
+ | into the central carbon metabolic pathway of <i>E. coli</i>. | ||
+ | PRK catalyzes the conversion of ribulose-5-phosphate (Ru5P) from the pentose phosphate pathway of | ||
+ | the central carbon metabolism to ribulose-1,5-biphosphate (RuBP). | ||
+ | One ATP is required for this conversion. | ||
+ | The accumulation of RuBP in <i>E.coli</i> would cause cell growth arrest because <i>E. coli</i> can not metabolize RuBP. | ||
+ | </p> | ||
+ | <img class="gif" src="https://static.igem.org/mediawiki/2018/7/71/T--NCKU_Tainan--design_PRK.gif" alt="PRK"> | ||
+ | |||
+ | <div class="row"> | ||
+ | <a class="btn col-md-12" data-toggle="collapse" href="#PRK_how_to_construct" role="button" aria-expanded="false" aria-controls="multiCollapseExample1"> | ||
+ | How do we construct this part? | ||
+ | <i class="fa fa-arrow-down fa-10" aria-hidden="true"></i> | ||
+ | </a> | ||
+ | </div> | ||
+ | <div class="collapse multi-collapse" id="PRK_how_to_construct"> | ||
+ | <div class="card card-body"> | ||
+ | <p class="pcontent">Steps involved in expressing PRK in <i>E. coli.</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. | ||
+ | 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. | ||
+ | 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. | ||
+ | </p> | ||
+ | </div> | ||
+ | </div> | ||
+ | <img class="bigimg" src="https://static.igem.org/mediawiki/2018/d/dd/T--NCKU_Tainan--design_PRK_construction.png" alt="PRK construction picture"> | ||
+ | |||
+ | <h5 class="question">How do we test its function?</h5> | ||
+ | <p class="pcontent">We initially decided to measure the concentration of RuBP by HPLC. | ||
+ | Our instructors pointed out some difficulties in HPLC measurement such as excessive noise signal in our sample. | ||
+ | We then designed an experiment to prove the function of PRK in an indirect manner: by measuring its growth rate. | ||
+ | RuBP, the product of PRK, is toxic to <i>E. coli</i>. | ||
+ | We expressed this protein independently in xylose M9 to check cell growth. | ||
+ | If the cell growth is arrested, we can indirectly conclude the function of PRK. | ||
+ | </p> | ||
+ | </div> | ||
+ | <div id="Rubisco"> | ||
+ | <h3>Rubisco</h3> | ||
+ | <h5 class="question">What is its function?</h5> | ||
+ | <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. | ||
+ | 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>. | ||
+ | After mining information from various publications, | ||
+ | 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. | ||
+ | Previous research has utilized <i>E. coli</i> as a host of random mutagenesis to enhance the activity of <i>Synechococcus</i> Rubisco. | ||
+ | </p> | ||
+ | <img class="gif" src="https://static.igem.org/mediawiki/2018/8/85/T--NCKU_Tainan--design_Rubisco.gif" alt="Rubisco"> | ||
+ | |||
+ | <div class="row"> | ||
+ | <a class="btn col-md-12" data-toggle="collapse" href="#RuBisCO_how_to_construct" role="button" aria-expanded="false" aria-controls="multiCollapseExample1"> | ||
+ | How do we construct this part? | ||
+ | <i class="fa fa-arrow-down fa-10" aria-hidden="true"></i> | ||
+ | </a> | ||
+ | </div> | ||
+ | <div class="collapse multi-collapse" id="RuBisCO_how_to_construct"> | ||
+ | <div class="card card-body"> | ||
+ | <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. | ||
+ | The sequence and the size of <i>rbcL</i> is much larger than other subunit, | ||
+ | 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 <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, | ||
+ | we would like to test various promoter combination to find out the most efficient combination for our pathway. | ||
+ | </p> | ||
+ | </div> | ||
+ | </div> | ||
+ | <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> | ||
+ | <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. | ||
+ | We then decided to determine its function by a total solution test which we will mention below. | ||
+ | </p> | ||
+ | </div> | ||
+ | <div id="CA"> | ||
+ | <h3>CA</h3> | ||
+ | <h5 class="question">What is its function?</h5> | ||
+ | <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. | ||
+ | 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 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. | ||
+ | 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> PCC 7002. | ||
+ | </p> | ||
+ | <img class="gif" src="https://static.igem.org/mediawiki/2018/3/34/T--NCKU_Tainan--design_CA.gif" alt="Rubisco"> | ||
+ | <div class="row"> | ||
+ | <a class="btn col-md-12" data-toggle="collapse" href="#CA_how_to_construct" role="button" aria-expanded="false" aria-controls="multiCollapseExample1"> | ||
+ | How do we construct this part? | ||
+ | <i class="fa fa-arrow-down fa-10" aria-hidden="true"></i> | ||
+ | </a> | ||
+ | </div> | ||
+ | <div class="collapse multi-collapse" id="CA_how_to_construct"> | ||
+ | <div class="card card-body"> | ||
+ | <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 | ||
+ | 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. | ||
+ | </p> | ||
+ | </div> | ||
+ | </div> | ||
+ | <img class="bigimg" src="https://static.igem.org/mediawiki/2018/7/78/T--NCKU_Tainan--design_CA_construction.png" alt="CA Construction picture"> | ||
+ | <h5 class="question">How do we test its function?</h5> | ||
+ | <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 | ||
+ | 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. | ||
+ | </p> | ||
+ | </div> | ||
+ | <div id="dual_plasmid_system"> | ||
+ | <h3>Dual Plasmid System</h3> | ||
+ | <p class="pcontent">We decided to construct the whole pathway with the dual plasmid system. | ||
+ | Previously, every basic part was the backbone conserved in the backbone of pSB1C3. | ||
+ | We then link the construction together and | ||
+ | even change the backbone of some composite parts to pSB3K3 for a lower protein expression. | ||
+ | </p> | ||
+ | <h5 class="question">Rubisco whole protein in pSB1C3</h5> | ||
+ | <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. | ||
+ | The former, the insert, was digested with EcoRI and SpeI and the latter, the backbone, | ||
+ | is digested with EcoRI and XbaI. | ||
+ | We ligate the backbone with the insert to complete this composite part. | ||
+ | </p> | ||
+ | <h5 class="question"><i>prk</i> gene into pSB3K3</h5> | ||
+ | <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>. | ||
+ | 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, | ||
+ | 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. | ||
+ | </p> | ||
+ | <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 <i>ccaA</i> and <i>prk</i>. | ||
+ | We construct it using the method mentioned in <i>rbc</i> whole construction. | ||
+ | We cloned the fragments into pSB3K3 for lower expression of PRK. | ||
+ | </p> | ||
+ | <h5 class="question">Transformation</h5> | ||
+ | <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). | ||
+ | Since BL21 (DE3) and W3110 (L5T7) contains T7 polymerase, | ||
+ | we co-transformed composite parts that contain T7 promoter into these strains. | ||
+ | We co-transform plasmid that only contains LacI promoter into W3110. | ||
+ | </p> | ||
+ | <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. | ||
+ | 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. | ||
+ | We then measure the optical intensity (O.D. 600) to characterize the cell growth. At a fixed time interval, | ||
+ | we use DNS assay to measure the sugar consumption of the bacteria. | ||
+ | By comparing the experimental group to the control group, | ||
+ | we can prove that our engineered strain utilize carbon dioxide as its carbon source. | ||
+ | </p> | ||
+ | </div> | ||
+ | <div id="pH_sensing_system"> | ||
+ | <h3>pH sensing system</h3> | ||
+ | <p class="pcontent">The pH sensing system, our side project, | ||
+ | is a system that allows us to monitor the pH in the surrounding medium | ||
+ | in our device at any time by observing the color change of the medium. | ||
+ | </p> | ||
+ | <p class="pcontent">We selected two pH sensitive promoter from <i>E. coli</i>: | ||
+ | P<sub>asr</sub> and P<sub>gadA</sub>. P<sub>gadA</sub> will be induced under neutral condition while P<sub>asr</sub> will be induced under acidic condition. | ||
+ | We cloned a GFP and sfGFP gene downstream of these promoters respectively, whose product will express green fluorescence once the promoter has been activated. For the design of P<sub>gadA</sub> sensing system, we took the previous constructed P<sub>gadA</sub> biobrick <a href="http://parts.igem.org/Part:BBa_K1962013" | ||
+ | style="color:#28ff28;">BBa_K1962013</a> from <a href="https://2016.igem.org/Team:Dundee" | ||
+ | style="color:#28ff28;">2016 iGEM Dundee team</a> as our reference. We also improve the P<sub>gadA</sub> biobrick to enhance the expression of GFP. | ||
+ | </p> | ||
+ | <p class="pcontent">In conclusion, when the color of the medium turns from turbid yellow to green, | ||
+ | it indicates the pH of the medium has altered so we can determine the pH condition of the medium. | ||
+ | </p> | ||
+ | <img class="gif" src="https://static.igem.org/mediawiki/2018/8/8e/T--NCKU_Tainan--design_pHsensor.gif" alt="pH"> | ||
+ | <div class="row"> | ||
+ | <a class="btn col-md-12" data-toggle="collapse" href="#pH_how_to_construct" role="button" aria-expanded="false" aria-controls="multiCollapseExample1"> | ||
+ | How do we construct this part? | ||
+ | <i class="fa fa-arrow-down fa-10" aria-hidden="true"></i> | ||
+ | </a> | ||
+ | </div> | ||
+ | <div class="collapse multi-collapse" id="pH_how_to_construct"> | ||
+ | <div class="card card-body"> | ||
+ | <p class="pcontent">We first extracted whole genome DNA from <i>E. coli</i> MG1655 and amplify both promoters by PCR | ||
+ | using primers that contains HindIII and SpeI. | ||
+ | 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 then transformed the plasmid into BL21 (DE3) to test its function. | ||
+ | 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. | ||
+ | </p> | ||
+ | </div> | ||
+ | </div> | ||
+ | <img class="bigimg" src="https://static.igem.org/mediawiki/2018/d/d2/T--NCKU_Tainan--design_pH_construction.png" alt="pH alert system construction picture"> | ||
+ | <h5 class="question">How do we determine its function?</h5> | ||
+ | <p class="pcontent">We measure the fluorescence intensity of the plasmid in different pH environment to | ||
+ | determine its promoter activity. We incubate the bacteria in pH modified M9 medium and | ||
+ | measure the fluorescence intensity (absorbance: 480 nm, excitation: 510 nm). | ||
+ | </p> | ||
+ | </div> | ||
+ | <div id="Reference"> | ||
+ | <h3>References</h3> | ||
+ | <ol> | ||
+ | <li class="smallp">Z. Cai, G. Liu, J. Zhang, Protein Cell (2014) 5: 552.</li> | ||
+ | <li class="smallp">F. Gong, “Quantitative Analysis of an Engineered CO<sub>2</sub> -Fixing <i>Escherichia Coli</i> Reveals Great Potential of Heterotrophic CO<sub>2</sub> Fixation.” Biotechnology for Biofuels, BioMed Central, 18 June 2015</li> | ||
+ | <li class="smallp">“The Coupling of Glycolysis and the Rubisco-Based Pathway through the Non-Oxidative Pentose Phosphate Pathway to Achieve Low Carbon Dioxide Emission Fermentation.” NeuroImage, Academic Press, 25 Mar. 2015</li> | ||
+ | <li class="smallp">“Sugar Synthesis from CO<sub>2</sub> in <i>Escherichia Coli</i>.” NeuroImage, Academic Press, 23 June 2016</li> | ||
+ | <li class="smallp">H. Cheng, E. J. Yang, Y. L. Liu, F. Y.Chenm, Y. Ou, S. Y. Li. “The Comprehensive Profile of Fermentation Products during in Situ CO<sub>2</sub> Recycling by Rubisco-Based Engineered <i>Escherichia Coli</i>.” Microbial Cell Factories, BioMed Central, 2 Aug. 2016</li> | ||
+ | </ol> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | <script> | ||
+ | $(document).ready(function() { | ||
+ | $(window).scroll(function() { | ||
+ | var scrollPercentage = (document.documentElement.scrollTop + document.body.scrollTop) / (document.documentElement.scrollHeight - document.documentElement.clientHeight); | ||
+ | if(scrollPercentage >= 0.95) { | ||
+ | var position = $("#sidelist").position(); | ||
+ | if(position == undefined){} | ||
+ | else{ | ||
+ | $('#sidelist').css({"position": "fixed", "top": "85px"}); | ||
+ | } | ||
+ | } else { | ||
+ | if ($(this).scrollTop() >= 500) { | ||
+ | var position = $("#sidelist").position(); | ||
+ | if(position == undefined){} | ||
+ | else{ | ||
+ | $('#sidelist').css({"position": "fixed", "top": "145px", "margin-top": "0px"}); | ||
+ | } | ||
+ | } else { | ||
+ | $('#sidelist').removeAttr('style'); | ||
+ | } | ||
+ | } | ||
+ | }); | ||
+ | $(function(){ | ||
+ | $('i.fa-arrow-up').click(function(){ | ||
+ | $('html, body').animate({scrollTop:0},600); | ||
+ | return false; | ||
+ | }); | ||
+ | }); | ||
+ | }); | ||
+ | </script> | ||
+ | <script src="https://2018.igem.org/Team:NCKU_Tainan/js/frame/T--NCKU_Tainan--jquery-1_12_4_min_js?action=raw&ctype=text/javascript"></script> | ||
+ | <script src="https://2018.igem.org/Template:NCKU_Tainan/js/bootstrap_min_js?action=raw&ctype=text/javascript"></script> | ||
+ | </body> | ||
+ | </html> | ||
+ | {{NCKU_Tainan/footer}} |
Latest revision as of 15:18, 3 November 2018