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

 
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     <!--Page_Content-->
 
     <!--Page_Content-->
 
     <div class="container content">
 
     <div class="container content">
        <h1 class="head">Results</h1>
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            <div class="headstyle">
 +
                <h1 class="head">Results</h1>
 +
            </div>
 +
            <div class="righttitle">
 +
                <h6 class="subtitle">Hard Work Pays Off</h6>
 +
            </div>
 
         <div class="navbar-example">
 
         <div class="navbar-example">
 
             <div class="row">
 
             <div class="row">
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                     <div data-spy="scroll" data-target="#sidelist" data-offset="0" class="scrollspy-example">
 
                     <div data-spy="scroll" data-target="#sidelist" data-offset="0" class="scrollspy-example">
 
                         <div class="container">
 
                         <div class="container">
 
 
 
                             <div id="Overview">
 
                             <div id="Overview">
                                </br></br></br></br>
 
 
                                 <h3>Overview of the result</h3>
 
                                 <h3>Overview of the result</h3>
 
                                 <ol>
 
                                 <ol>
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                             <div id="Construction">
 
                             <div id="Construction">
                                </br></br></br></br>
 
 
                                 <h3>Construction and functional test </h3></br>
 
                                 <h3>Construction and functional test </h3></br>
  
 
                                 <div id="pt">
 
                                 <div id="pt">
                                     <h8>PRK</h8></br></br>
+
                                     <h8>PRK (PHOSPHORIBULOKINASE)</h8></br></br>
 
                                     <p class="pcontent">
 
                                     <p class="pcontent">
 
                                         Achievements:</br>
 
                                         Achievements:</br>
Line 65: Line 66:
 
                                         <li>PRK toxicity test proves that the function of it varies when cloned
 
                                         <li>PRK toxicity test proves that the function of it varies when cloned
 
                                             into
 
                                             into
                                             different plasmid</li>
+
                                             different plasmids</li>
 
                                     </ol></br>
 
                                     </ol></br>
  
 
                                     <p class="pcontent">
 
                                     <p class="pcontent">
                                         We constructed PRK fragments (<a href="http://parts.igem.org/Part:BBa_K2762007"
+
                                         We constructed <i>prk</i> fragments (<a href="http://parts.igem.org/Part:BBa_K2762007"
 
                                             style="color:#28ff28;">BBa_K2762007</a>) from IDT DNA synthesis. After PCR
 
                                             style="color:#28ff28;">BBa_K2762007</a>) from IDT DNA synthesis. After PCR
 
                                         amplification,
 
                                         amplification,
                                         PRK is then cloned into pSB1C3 and transformed into DH5 alpha. SDS-PAGE ensured that
+
                                         <i>prk</i> is then cloned into pSB1C3 and transformed into DH5 alpha. SDS-PAGE ensured that
 
                                         the protein expression was as expected. The results are shown below:
 
                                         the protein expression was as expected. The results are shown below:
 
                                     </p>
 
                                     </p>
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                                         inside the cell. Our instructors pointed out some difficulties in HPLC
 
                                         inside the cell. Our instructors pointed out some difficulties in HPLC
 
                                         measurement such as excessive noise signal in our sample. We, therefore,
 
                                         measurement such as excessive noise signal in our sample. We, therefore,
                                         determined to test its function with a toxicity test. The product of PRK-RuBP
+
                                         determined to test its function with a toxicity test. The product of PRK, RuBP,
                                         cannot be metabolite by wild-type <i>E. coli</i>. The accumulation of RuBP
+
                                         cannot be metabolized by wild-type <i>E. coli</i>. The accumulation of RuBP
 
                                         depletes
 
                                         depletes
 
                                         the sugar from the native pentose phosphate pathway. Lack of carbon source, the
 
                                         the sugar from the native pentose phosphate pathway. Lack of carbon source, the
 
                                         growth of that strain may be repressed. We incubate the PRK expressing strain
 
                                         growth of that strain may be repressed. We incubate the PRK expressing strain
 
                                         and control stain that contains no plasmid in M9 medium and altered M9 medium
 
                                         and control stain that contains no plasmid in M9 medium and altered M9 medium
                                         with 0.4% xylose as its sole carbon source. In normal M9 medium, glucose will
+
                                         with 4 (g/l) xylose as its sole carbon source. In normal M9 medium, glucose will
 
                                         not be converted into RuBP. In altered M9 medium, xylose will go through the
 
                                         not be converted into RuBP. In altered M9 medium, xylose will go through the
 
                                         native pathway and be converted into RuBP. Growth arrest of PRK strain should
 
                                         native pathway and be converted into RuBP. Growth arrest of PRK strain should
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                                     <p class="pcontent">
 
                                     <p class="pcontent">
                                         Fig 4. Compares the growth in M9 xylose medium of PRK expressing strain in
+
                                         Fig 4. Compares the growth in M9 xylose medium of PRK expressing strain which prk is cloned into
 
                                         high
 
                                         high
                                         and low copy number plasmid. The low copy number plasmid, pSB3K3, shows a
+
                                         and low copy number plasmids respectively. The low copy number plasmid, pSB3K3, shows a
                                         little bit of growth retard compare to non-PRK expressing strain. However, the
+
                                         little bit of growth retard compare to the control strain. However, its
                                         toxicity is much less than high copy number expressing strain.
+
                                         toxicity is much less than that expressed in high copy number.
 
                                     </p></br>
 
                                     </p></br>
  
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                                         We also transformed pSB3K3-<i>prk</i> into W3110 strain. W3110 is reported to have
 
                                         We also transformed pSB3K3-<i>prk</i> into W3110 strain. W3110 is reported to have
 
                                         higher pressure tolerance. The trend of the results is similar to that of the
 
                                         higher pressure tolerance. The trend of the results is similar to that of the
                                         BL21 (DE3) but there is no statistically significant between the experiment and
+
                                         BL21 (DE3), but the difference between experiement and control group is not obvious                            . We deduce that PRK can still function in W3110 since the
                                        the control group. We deduce that PRK can still function in W3110 since the
+
 
                                         trend matches our expectation. As pSB3K3 is a low copy number plasmid, the
 
                                         trend matches our expectation. As pSB3K3 is a low copy number plasmid, the
 
                                         expression of the protein may be lower than that of high copy number plasmid.
 
                                         expression of the protein may be lower than that of high copy number plasmid.
 
                                         The
 
                                         The
                                         pressure tolerance of W3110 strain may also lessen the toxicity influence by
+
                                         pressure tolerance of W3110 strain may also lessen the influence of toxicity by
 
                                         PRK.
 
                                         PRK.
 
                                     </p>
 
                                     </p>
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                                     <img class="contentimg fig5" src="https://static.igem.org/mediawiki/2018/f/f6/T--NCKU_Tainan--Results_fig_4_2.PNG">
 
                                     <img class="contentimg fig5" src="https://static.igem.org/mediawiki/2018/f/f6/T--NCKU_Tainan--Results_fig_4_2.PNG">
  
                                     <p class="pcontent">
+
                                     <p class="pcenter">
 
                                         Fig 5. The result of PRK test in W3110
 
                                         Fig 5. The result of PRK test in W3110
 
                                     </p></br>
 
                                     </p></br>
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                                 <div id="pt">
 
                                 <div id="pt">
                                     <h8>CA</h8></br></br>
+
                                     <h8>CA (Carbonic Anhydrase)</h8></br></br>
 
                                     <p class="pcontent">
 
                                     <p class="pcontent">
 
                                         Achievements:</br>
 
                                         Achievements:</br>
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                                         and the time course (sec) of pH decrease from 8.3 to 6.3 was recorded. CA
 
                                         and the time course (sec) of pH decrease from 8.3 to 6.3 was recorded. CA
 
                                         activity was calculated using a Wilbur–Anderson unit (WAU) per milliliter of
 
                                         activity was calculated using a Wilbur–Anderson unit (WAU) per milliliter of
                                         sample. The definition for WAU is (T<sub>0</sub>-T)/(T<sub>0</sub>) in which T<sub>0</sub>
+
                                         sample. The definition for WAU is (T<sub>0</sub>-T)/ (T<sub>0</sub>) in which T<sub>0</sub>
 
                                         and T was the time required for the pH drop from 8.3 to 6.3, with and without
 
                                         and T was the time required for the pH drop from 8.3 to 6.3, with and without
 
                                         CA, respectively. The enzyme activity of our CA is 21.8
 
                                         CA, respectively. The enzyme activity of our CA is 21.8
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                                         Fig 8. Confirmation of <i>rbcX</i> and <i>rbcS</i> digestion
 
                                         Fig 8. Confirmation of <i>rbcX</i> and <i>rbcS</i> digestion
  
                                         Fig 9.  Confirmation of RbcX and RbcS expression in BL21 (DE3) The expected protein
+
                                         Fig 9.  Confirmation of <i>rbcX</i> and <i>rbcS</i> expression in BL21 (DE3). The expected protein
                                         size is 15.3 kDA and 13.8kDA respectively.
+
                                         size is 15.3 kDA and 13.8 kDA respectively.
 
                                     </p>
 
                                     </p>
  
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                                     <p class="pcontent">
 
                                     <p class="pcontent">
                                         Fig 10. Confirmation of rbcL digestion.</br>
+
                                         Fig 10. Confirmation of <i>rbcL</i> digestion.</br>
  
                                         Fig 11. Confirmation of rbcL expression in DH5 alpha. The expected protein size is
+
                                         Fig 11. Confirmation of <i>rbcL</i> expression in DH5 alpha. The expected protein size is
                                         52.37kDa.</br>
+
                                         52.37 kDa.</br>
 
                                     </p>
 
                                     </p>
 
+
<br>
 
                                     <p class="pcontent">
 
                                     <p class="pcontent">
 
                                         After mining a lot of information from the publications, we found out a method
 
                                         After mining a lot of information from the publications, we found out a method
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                             <div id="Total_solution">
 
                             <div id="Total_solution">
                                </br></br></br></br>
 
 
                                 <h3>Total solution</h3>
 
                                 <h3>Total solution</h3>
  
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                                                 previous researches determine the efficiency of carbon fixation via measuring
 
                                                 previous researches determine the efficiency of carbon fixation via measuring
 
                                                 the decrease of carbon dioxide concentration in the closed system or measure
 
                                                 the decrease of carbon dioxide concentration in the closed system or measure
                                                 the weight
+
                                                 the weight percentage of <sup>14</sup>C radioisotope in the dry cell. However, due to biosafety
                                                percentage of C14 radioisotope in the dry cell. However, due to biosafety
+
 
                                                 constrain of our lab, we can barely use the radioisotope. Measuring the
 
                                                 constrain of our lab, we can barely use the radioisotope. Measuring the
 
                                                 decrease of carbon dioxide concentration in the closed system is also
 
                                                 decrease of carbon dioxide concentration in the closed system is also
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                                                 density
 
                                                 density
 
                                                 O.D. 600 and xylose consumption. We have measure various construction to prove
 
                                                 O.D. 600 and xylose consumption. We have measure various construction to prove
                                                 that the enzyme of our construction is necessary for carbon fixation.
+
                                                 that all the enzymes in our design is necessary for carbon fixation.
 
                                             </p></br>
 
                                             </p></br>
  
 
                                             <p class="pcontent">
 
                                             <p class="pcontent">
                                                 The test samples below were incubated in an altered M9 medium which substitutes
+
                                                 The bacteria samples in total solution test were incubated in an altered M9 medium which substitutes
                                                 glucose to xylose. 1/1000 of LB medium was added to support some rare elements.
+
                                                 glucose to xylose. 1/1000 of LB medium was added to support the trace elements.
 
                                                 Since the concentration of LB medium is too low, it doesn’t contribute the
 
                                                 Since the concentration of LB medium is too low, it doesn’t contribute the
 
                                                 carbon source of the bacteria.
 
                                                 carbon source of the bacteria.
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                                                 We defined a new index, Xylose Utilization Index, to describe the potential of
 
                                                 We defined a new index, Xylose Utilization Index, to describe the potential of
 
                                                 carbon fixation. We can compare this index of each strain to find out the
 
                                                 carbon fixation. We can compare this index of each strain to find out the
                                                 strain that has the highest capacity of carbon fixing.
+
                                                 strain that has the highest capacity of carbon fixation.
 
                                             </p></br>
 
                                             </p></br>
  
 
                                             <p class="pcontent">
 
                                             <p class="pcontent">
                                                 To define the XUI index, we firstly made two assumptions:
+
                                                 To define the XUI, we firstly made two assumptions:
 
                                             </p>
 
                                             </p>
  
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                                             <ol start="2">
 
                                             <ol start="2">
 
                                                 <li>The elemental formula of <i>E. coli</i> should be fixed or varies within a
 
                                                 <li>The elemental formula of <i>E. coli</i> should be fixed or varies within a
                                                     small range. Although there may exist slightly different in different
+
                                                     small range. Although the formula may have variations in different
 
                                                     growth condition, we assume that such error can be ignored during the
 
                                                     growth condition, we assume that such error can be ignored during the
 
                                                     following calculation.</li>
 
                                                     following calculation.</li>
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                                             <p class="pcontent">
 
                                             <p class="pcontent">
                                                 After these two assumptions, the Xylose Consumption Index is designed to
+
                                                 After these two assumptions, the Xylose Utilization Index is designed to
 
                                                 evaluate
 
                                                 evaluate
 
                                                 the carbon fixation ability of each strain. The definition of the index is
 
                                                 the carbon fixation ability of each strain. The definition of the index is
Line 383: Line 381:
 
                                                 producing the same amount of carbon biomass, it requires less xylose. We can
 
                                                 producing the same amount of carbon biomass, it requires less xylose. We can
 
                                                 thus
 
                                                 thus
                                                 compare the XUI of each strain to determine the possible strain that fixes
+
                                                 compare the XUI of each strain to determine the strain that fixes
 
                                                 carbon.
 
                                                 carbon.
 
                                                 The less the XUI in the sample, the more possibility that it fixes carbon.
 
                                                 The less the XUI in the sample, the more possibility that it fixes carbon.
 
                                             </p>
 
                                             </p>
 
+
                                            <p class="pcontent">$${XUI = {{xylose \  consumption \ (g/l)} \over {O.D. 600}}}$$</p>
 
                                             <img class="contentimg" src="https://static.igem.org/mediawiki/2018/1/1b/T--NCKU_Tainan--CO2_results.gif">
 
                                             <img class="contentimg" src="https://static.igem.org/mediawiki/2018/1/1b/T--NCKU_Tainan--CO2_results.gif">
  
Line 418: Line 416:
  
 
                                             <p class="pcontent">
 
                                             <p class="pcontent">
                                                 Fig 14. shows the growth of engineered (contains Rubisco and PRK) W3110 (L5T7), BL21(DE3), W3110 incubated in normal
+
                                                 Fig 14. shows the growth of engineered (contains Rubisco and PRK) W3110 (L5T7), BL21 (DE3), W3110 incubated in normal
 
                                                 incubator for 24 hours. The growth of W3110 (L5T7) is not obvious while other
 
                                                 incubator for 24 hours. The growth of W3110 (L5T7) is not obvious while other
 
                                                 strains show growth after 24hours.
 
                                                 strains show growth after 24hours.
Line 435: Line 433:
 
                                             style="color:#28ff28;">BBa_K2762007</a>) was cloned into pSB3K3. Both
 
                                             style="color:#28ff28;">BBa_K2762007</a>) was cloned into pSB3K3. Both
 
                                         plasmids were then co-transformed into BL21 (DE3). We
 
                                         plasmids were then co-transformed into BL21 (DE3). We
                                         measure the XUI of the strain and compare to the control that IPTG was not
+
                                         measured the XUI of the strain and compared them with the control group that IPTG was not
 
                                         added and BL21 (DE3) without plasmid. IPTG can induce the promoter
 
                                         added and BL21 (DE3) without plasmid. IPTG can induce the promoter
 
                                         P<sub>T7</sub> to produce the downstream enzyme. The growth of each strain is
 
                                         P<sub>T7</sub> to produce the downstream enzyme. The growth of each strain is
 
                                         first examined. The IPTG induced strain showed growth retard. We assume the
 
                                         first examined. The IPTG induced strain showed growth retard. We assume the
 
                                         cause of growth retard is due to the pressure from overexpressing the protein
 
                                         cause of growth retard is due to the pressure from overexpressing the protein
                                         rubisco. The control strain without IPTG induction produce less rubisco enzyme
+
                                         Rubisco. The control strain without IPTG induction produce less Rubisco enzyme
                                         than the experiment and had less pressure. We then compare the XUI of each
+
                                         than the experiment and has less pressure. We then compare the XUI of each
 
                                         strain and discovered that control strain without IPTG induction produces less
 
                                         strain and discovered that control strain without IPTG induction produces less
                                         rubisco enzyme than the experiment. Without rubisco, the bypass pathway is not
+
                                         Rubisco enzyme than the experiment. Without Rubisco, the bypass pathway is not
 
                                         capable of using CO<sub>2</sub>. We found out that the strain without Rubisco
 
                                         capable of using CO<sub>2</sub>. We found out that the strain without Rubisco
 
                                         has higher
 
                                         has higher
                                         XUI, symbolizing that rubisco is essential in carbon fixation pathway.
+
                                         XUI, symbolizing that Rubisco is essential in the carbon fixation pathway.
 
                                     </p>
 
                                     </p>
  
Line 454: Line 452:
  
 
                                     <p class="pcontent">
 
                                     <p class="pcontent">
                                         Fig 15. Shows the growth and XUI measured in 5% CO<sub>2</sub> incubation of 12
+
                                         Fig 15. Shows the growth and XUI measured in 5% CO<sub>2</sub> incubation for 12
 
                                         hours
 
                                         hours
                                         respectively. Lower growth of the strain that contains. The XUI of the strain
+
                                         respectively. The strain that contains PRK and Rubisco shows little growth. The XUI of the strain
 
                                         that contains both Rubisco and PRK shows statistically significant decrease
 
                                         that contains both Rubisco and PRK shows statistically significant decrease
 
                                         compare to strain without both enzymes.</br>
 
                                         compare to strain without both enzymes.</br>
Line 510: Line 508:
 
                                         the difference of XUI between two <i>E. coli</i> strain. We found out that both
 
                                         the difference of XUI between two <i>E. coli</i> strain. We found out that both
 
                                         strain
 
                                         strain
                                         shows similar trend: the XUI will be lower with the expression of the constructed protein. HoweverW3110 has a higher XUI compared with BL21 (DE3) in
+
                                         shows similar trend: the XUI will be lower with the expression of the constructed protein. However, W3110 has a higher XUI compared with BL21 (DE3) in
 
                                         constructed strain as well as the strain without plasmid. We infer two reasons
 
                                         constructed strain as well as the strain without plasmid. We infer two reasons
 
                                         that cause the difference of XUI:
 
                                         that cause the difference of XUI:
Line 516: Line 514:
  
 
                                     <ol>
 
                                     <ol>
                                         <li>W3110 “wildtype” strain has more flexible metabolic network but consumes
+
                                         <li>W3110 “wildtype” strain has more flexible metabolic network. The carbon flux to pentose phosphate pathway of W3110 is more than that of BL21 (DE3) and thus consumes
 
                                             more xylose compare to lab strains such as BL21 (DE3).</li>
 
                                             more xylose compare to lab strains such as BL21 (DE3).</li>
  
 
                                         <li>The constructed protein expression in W3110 may be less than BL21 (DE3) lab
 
                                         <li>The constructed protein expression in W3110 may be less than BL21 (DE3) lab
                                             strain. BL21 (DE3) commonly used to express protein. We inferred that with
+
                                             strain. BL21 (DE3) is commonly used to express protein. We inferred that with
 
                                             more protein been expressed, the bypass pathway in BL21 (DE3) will be more
 
                                             more protein been expressed, the bypass pathway in BL21 (DE3) will be more
 
                                             favored than the W3110 strain.</li>
 
                                             favored than the W3110 strain.</li>
Line 542: Line 540:
 
                                     <p class="pcontent">
 
                                     <p class="pcontent">
 
                                         Finally, we compare the XUI under different CO<sub>2</sub> concentration. We
 
                                         Finally, we compare the XUI under different CO<sub>2</sub> concentration. We
                                         incubated the
+
                                         incubated the engineered
 
                                         bacteria in normal incubator without CO<sub>2</sub> input and the cell culture
 
                                         bacteria in normal incubator without CO<sub>2</sub> input and the cell culture
 
                                         incubator
 
                                         incubator
Line 561: Line 559:
 
                                         Fig 18. The comparison of the growth and the XUI of the BL21 (DE3) that contains
 
                                         Fig 18. The comparison of the growth and the XUI of the BL21 (DE3) that contains
 
                                         all three enzymes in normal incubator and 5% CO<sub>2</sub> incubator. The
 
                                         all three enzymes in normal incubator and 5% CO<sub>2</sub> incubator. The
                                         strain was grown in
+
                                         strain grown in
 
                                         CO<sub>2</sub> incubator showed better growth and lower XUI, which indicates
 
                                         CO<sub>2</sub> incubator showed better growth and lower XUI, which indicates
 
                                         that our
 
                                         that our
Line 574: Line 572:
  
 
                             <div id="Carbon_fixation">
 
                             <div id="Carbon_fixation">
 +
                                <h3>Carbon fixation</h3>
 
                                 <div class="row">
 
                                 <div class="row">
 
                                     <a class="btn col-md-12" data-toggle="collapse" href="#Estimation_Carbon_fixation" role="button" aria-expanded="false" aria-controls="multiCollapseExample1">
 
                                     <a class="btn col-md-12" data-toggle="collapse" href="#Estimation_Carbon_fixation" role="button" aria-expanded="false" aria-controls="multiCollapseExample1">
                                         Estimation of the amount of the carbon fixation
+
                                         Amount of Carbon Fixed: 0.575 mg / l * hr
 
                                         <i class="fa fa-arrow-down fa-10" aria-hidden="true"></i>
 
                                         <i class="fa fa-arrow-down fa-10" aria-hidden="true"></i>
 
                                     </a>
 
                                     </a>
Line 592: Line 591:
  
 
                                                     $${\{Input\ to\ the\ system\}\ –\ \{Output\ to\ the\ system\}\ =\
 
                                                     $${\{Input\ to\ the\ system\}\ –\ \{Output\ to\ the\ system\}\ =\
                                                     \{Accumulation\ in\ the\ syste\}}$$
+
                                                     \{Accumulation\ in\ the\ system\}}$$
  
 
                                                     A system can be defined as an arbitrary portion of a process considered for
 
                                                     A system can be defined as an arbitrary portion of a process considered for
Line 603: Line 602:
 
                                         <div id="pt">
 
                                         <div id="pt">
 
                                             <p class="pcontent">
 
                                             <p class="pcontent">
                                                     The engineered <i>E. coli</i> BL21 (DE3) are cultured in M9 medium with formula
+
                                                     The engineered <i>E. coli</i> BL21 (DE3) is cultured in M9 medium with formula
 
                                                     adjusted so that xylose is the sole carbon source. The aforementioned M9 Medium
 
                                                     adjusted so that xylose is the sole carbon source. The aforementioned M9 Medium
 
                                                     contains
 
                                                     contains
                                                     0.4% xylose and 1/1000 LB medium (the carbon consumed from LB medium can be
+
                                                     4 (g/l) xylose and 1/1000 LB medium (the carbon consumed from LB medium can be
 
                                                     ignored). By applying the law of conservation of mass, which states that mass
 
                                                     ignored). By applying the law of conservation of mass, which states that mass
 
                                                     may neither be created nor destroyed, the material balance for carbon in an
 
                                                     may neither be created nor destroyed, the material balance for carbon in an
Line 646: Line 645:
 
                                                     mass.
 
                                                     mass.
  
                                                     $${C_{biomass}\ =\ 0.45\ ×\ 0.35\ ×\ 48\%}$$
+
                                                     $${C_{biomass}\ =\ 0.4511\ ×\ 0.35\ ×\ 48\%}$$
                                                     $${=\ 0.0756\ g/L}$$
+
                                                     $${=\ 0.0758\ g/L}$$
 
                                             </p>
 
                                             </p>
  
Line 655: Line 654:
 
                                                     xylose consumed per unit volume of broth to the mass percent of carbon in
 
                                                     xylose consumed per unit volume of broth to the mass percent of carbon in
 
                                                     xylose. Xylose consumption is calculated by using a DNS kit that measures the
 
                                                     xylose. Xylose consumption is calculated by using a DNS kit that measures the
                                                     concentration of reducing sugar and the result obtained is 0.172324g of xylose
+
                                                     concentration of reducing sugar and the result obtained is 0.1723g of xylose
 
                                                     consumed per liter of M9 medium. Carbon mass percentage of xylose
 
                                                     consumed per liter of M9 medium. Carbon mass percentage of xylose
 
                                                     is 40%.
 
                                                     is 40%.
  
                                                     $${C_{xylose}\ =\ 0.172324\ ×\ 40\%\ =\ 0.0689296\ g/L}$$
+
                                                     $${C_{xylose}\ =\ 0.1723\ ×\ 40\%\ =\ 0.0689\ g/L}$$
  
 
                                                     By equation (3)
 
                                                     By equation (3)
  
                                                     $${C_{CO_2\ net}\ =\ 0.0756\ -\ 0.0689296}$$
+
                                                     $${C_{CO_2\ net}\ =\ 0.0758\ -\ 0.0689}$$
  
                                                     $${=\ 0.0066704\ g/L}$$
+
                                                     $${=\ 0.0069\ g/L}$$
  
 
                                                     Since the <i>E. coli</i> has been cultured for 12 hours, we can calculate the
 
                                                     Since the <i>E. coli</i> has been cultured for 12 hours, we can calculate the
Line 673: Line 672:
 
                                                     $${Rate\ of\ carbon\ fixation\ =\ {𝐶_{𝐶𝑂_2\ 𝑛𝑒𝑡}\over 12}}$$
 
                                                     $${Rate\ of\ carbon\ fixation\ =\ {𝐶_{𝐶𝑂_2\ 𝑛𝑒𝑡}\over 12}}$$
  
                                                     $${=\ {0.0066704\over 12}}$$
+
                                                     $${=\ {0.0069\over 12}}$$
  
                                                     $${=\ 0.5558\ {mg\over L ∙hr}}$$
+
                                                     $${=\ 0.575\ {mg\over L ∙hr}}$$
  
                                                     To find out how much carbon in biomass comes from the carbon in CO<sub>2</sub>
+
                                                     To find out how much carbon in biomass comes from the carbon in CO2 captured by the heterotrophic microbes, we can divide equation (3) by the mass percentage of carbon in biomass:
                                                    captured by
+
                                                    the heterotrophic microbes, divide the net amount of carbon fixed by the mass
+
                                                    percent of carbon in biomass.
+
  
                                                    $${Ratio\ of\ carbon\ in\ CO_2\ fixed\ to\ carbon\ in\ biomass\ =\
+
 
                                                    {0.0066704\over 0.0756}}$$
+
                                    </p>
                                                    $${=\ 8.82\%}$$
+
                                    <p class="pcontent">$${{{ \{ CO_{2 net}} \} \over \{ {C_{biomass}} \} } \geq {1 - 
 +
{ \{ {C_{xylose}} \} \over \{ {C_{biomass}} \} }}}$$</p>
 +
<p class="pcontent">We can thus calculate the ratio with our experiment results:</p>
 +
                                    <p class="pcontent">$${{Ratio \ of \ carbon \ in \ CO_2 \ fixed \ to \ carbon \ in \ biomass} = {1 -{0.0689 \over 0.0758}} = 9.1 \%}$$
 
                                             </p>
 
                                             </p>
 
                                         </div>
 
                                         </div>
Line 692: Line 691:
  
 
                             <div id="pH_Senesing">
 
                             <div id="pH_Senesing">
                                </br></br></br></br>
 
 
                                 <h3>pH sensing system</h3>
 
                                 <h3>pH sensing system</h3>
  
Line 707: Line 705:
 
                                     <li>Measure and characterize the long-term fluorescence intensity of P<sub>gadA</sub> (<a href="http://parts.igem.org/Part:BBa_K1962013"
 
                                     <li>Measure and characterize the long-term fluorescence intensity of P<sub>gadA</sub> (<a href="http://parts.igem.org/Part:BBa_K1962013"
 
                                             style="color:#28ff28;">BBa_K1962013</a>)</li>
 
                                             style="color:#28ff28;">BBa_K1962013</a>)</li>
 +
<li>Improve the previous biobrick of P<sub>gadA</sub> (<a href="http://parts.igem.org/Part:BBa_K1962013"
 +
                                            style="color:#28ff28;">BBa_K1962013</a>)</li>
 +
  
 
                                 </ol></br>
 
                                 </ol></br>
Line 718: Line 719:
 
                                         of it is small. For the construction of P<sub>asr</sub>, we cloned into the
 
                                         of it is small. For the construction of P<sub>asr</sub>, we cloned into the
 
                                         plasmid that
 
                                         plasmid that
                                         contains both rbc and GFP. For the construction of P<sub>gadA</sub>, we took
+
                                         contains both rbs and GFP. For the construction of P<sub>gadA</sub>, we took
 
                                         the
 
                                         the
 
                                         constructed part from 2016 Dundee iGEM team as a reference. Both parts were
 
                                         constructed part from 2016 Dundee iGEM team as a reference. Both parts were
Line 730: Line 731:
 
                                 <div id="pt">
 
                                 <div id="pt">
 
                                     <p class="pcontent">
 
                                     <p class="pcontent">
                                         P<sub>asr</sub> is reported to be induced in acidic condition. We think that it
+
                                         P<sub>asr</sub> (<a href="http://parts.igem.org/Part:BBa_K1231000"
 +
                                            style="color:#28ff28;">BBa_K1231000</a>) is reported to be induced in acidic condition. We think that it
 
                                         can be used
 
                                         can be used
 
                                         to report the abnormal acidity of the medium. We thus determine to measure the
 
                                         to report the abnormal acidity of the medium. We thus determine to measure the
Line 738: Line 740:
 
                                         modified with 1M HCl). We then took the sample and incubate in the 96 well and
 
                                         modified with 1M HCl). We then took the sample and incubate in the 96 well and
 
                                         measure its fluorescence intensity for every 3 minutes. We found out that the
 
                                         measure its fluorescence intensity for every 3 minutes. We found out that the
                                         promoter Pasr will be induced at the pH value below four within 30 minutes. The
+
                                         promoter P<sub>asr</sub> will be induced at the pH value below four within 30 minutes. The
 
                                         different fluorescence intensity can be observed within 30 minutes. The
 
                                         different fluorescence intensity can be observed within 30 minutes. The
 
                                         fluorescence had the peak at pH value of 4.25.
 
                                         fluorescence had the peak at pH value of 4.25.
Line 752: Line 754:
  
 
                                     <p class="pcontent">
 
                                     <p class="pcontent">
                                         Based on the data has shown above, we could conclude that Pasr is an acidic
+
                                         Based on the data has shown above, we could conclude that P<sub>asr</sub> is an acidic
 
                                         promoter as it has a high expression of fluorescence at pH 4.25 and pH 5. The
 
                                         promoter as it has a high expression of fluorescence at pH 4.25 and pH 5. The
                                         results show that Pasr constructed pH sensing system can be used as an alert.
+
                                         results show that P<sub>asr</sub> constructed pH sensing system can be used as an alert.
 
                                         When the medium turns acidic, fluorescence can be easily observed. We believe
 
                                         When the medium turns acidic, fluorescence can be easily observed. We believe
 
                                         that this system can also be applied to various bio-detection system.
 
                                         that this system can also be applied to various bio-detection system.
Line 763: Line 765:
  
 
                                     <p class="pcontent">
 
                                     <p class="pcontent">
                                         P<sub>gadA</sub> was previously reported to be induced under neutral and mild
+
                                         P<sub>gadA</sub> (<a href="http://parts.igem.org/Part:BBa_K1962013"
 +
                                            style="color:#28ff28;">BBa_K1962013</a>) was previously reported to be induced under neutral and mild
 
                                         acidic
 
                                         acidic
                                         environment. We measure the fluorescence intensity for 14 hours. We pre-cultured
+
                                         environment. We measured the fluorescence intensity for 14 hours. We pre-cultured
 
                                         the strain and incubate the strain with pH modified M9 medium (the pH value is
 
                                         the strain and incubate the strain with pH modified M9 medium (the pH value is
 
                                         modified with 1M HCl). The induction of P<sub>gadA</sub> is observed under
 
                                         modified with 1M HCl). The induction of P<sub>gadA</sub> is observed under
 
                                         neutral and
 
                                         neutral and
                                         mild expression.
+
                                         weak acidic environment.
 
                                     </p>
 
                                     </p>
  
Line 778: Line 781:
 
                                         excitation: 535 nm) expressed by P<sub>gadA</sub> in different pH.
 
                                         excitation: 535 nm) expressed by P<sub>gadA</sub> in different pH.
 
                                     </p>
 
                                     </p>
 +
<br>
 +
<h8>Improvement of P<sub>gadA</sub></h8></br></br>
  
 
                                     <p class="pcontent">
 
                                     <p class="pcontent">
Line 784: Line 789:
 
                                         P<sub>asr</sub> and would like to improve the sensitivity of this biobrick. We
 
                                         P<sub>asr</sub> and would like to improve the sensitivity of this biobrick. We
 
                                         thus add a
 
                                         thus add a
                                         riboJ sequence at the downstream of P<sub>gadA</sub>. For more information,
+
                                         RiboJ sequence at the downstream of P<sub>gadA</sub>. RiboJ sequence is reported to increase the expression of downstream protein. We thus compare the fluorescence of previous and improved biobrick. We discover that with RiboJ, the protein of down-stream reporter protein is increased.</p>
 +
 
 +
<img class="contentimg fig3" src="https://static.igem.org/mediawiki/2018/f/fd/T--NCKU_Tainan--PGADA_OMPARISON.png">
 +
 
 +
<p class="pcontent">
 +
                                        Fig 21. The data compares the fluorescence of P<sub>gadA</sub> and P<sub>gadA</sub> with RiboJ sequence.
 +
                                    </p>
 +
<br>
 +
<p class="pcontent">For more information,
 
                                         please check
 
                                         please check
 
                                         the <a href="https://2018.igem.org/Team:NCKU_Tainan/Improve"
 
                                         the <a href="https://2018.igem.org/Team:NCKU_Tainan/Improve"
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             $(window).scroll(function () {
                 if ($(this).scrollTop() >= 90) {
+
                 if ($(this).scrollTop() >= 500) {
 
                     var position = $("#sidelist").position();
 
                     var position = $("#sidelist").position();
 
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Latest revision as of 03:26, 18 October 2018

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