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<!--Page_Content--> | <!--Page_Content--> | ||
<div class="container content"> | <div class="container content"> | ||
− | + | <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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<a class="list-group-item list-group-item-action" href="#Total_solution">Total solution</a> | <a class="list-group-item list-group-item-action" href="#Total_solution">Total solution</a> | ||
<a class="list-group-item list-group-item-action" href="#Carbon_fixation">Carbon fixation</a> | <a class="list-group-item list-group-item-action" href="#Carbon_fixation">Carbon fixation</a> | ||
− | <a class="list-group-item list-group-item-action" href="#pH_Senesing">pH Sensing System</a> | + | <a class="list-group-item list-group-item-action" href="#pH_Senesing">pH Sensing System</a> |
+ | <a class="list-group-item list-group-item-action" href="#References">References</a> | ||
<a class="list-group-item list-group-item-action" href="#"><i class="fa fa-arrow-up fa-1x" | <a class="list-group-item list-group-item-action" href="#"><i class="fa fa-arrow-up fa-1x" | ||
aria-hidden="true"></i></a> | aria-hidden="true"></i></a> | ||
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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"> | ||
− | |||
<h3>Overview of the result</h3> | <h3>Overview of the result</h3> | ||
<ol> | <ol> | ||
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<li>Estimate the carbon fixation amount with our experiment result.</li> | <li>Estimate the carbon fixation amount with our experiment result.</li> | ||
<li>Characterize the pH sensing promoter P<sub>asr</sub> (<a href="http://parts.igem.org/Part:BBa_K1231000" | <li>Characterize the pH sensing promoter P<sub>asr</sub> (<a href="http://parts.igem.org/Part:BBa_K1231000" | ||
− | style="color:#28ff28;">BBa_K1231000</a>), and improve P<sub>gadA</sub> biobrick (<a href="http://parts.igem.org/Part:BBa_K1962013" | + | style="color:#28ff28;">BBa_K1231000</a>), and improve P<sub>gadA</sub> biobrick (<a href="http://parts.igem.org/Part:BBa_K1962013" |
style="color:#28ff28;">BBa_K1962013</a>).</li> | style="color:#28ff28;">BBa_K1962013</a>).</li> | ||
</ol> | </ol> | ||
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<div id="Construction"> | <div id="Construction"> | ||
− | |||
<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> | ||
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<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 | + | different plasmids</li> |
</ol></br> | </ol></br> | ||
<p class="pcontent"> | <p class="pcontent"> | ||
− | We constructed | + | 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, | ||
− | + | <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 | + | determined to test its function with a toxicity test. The product of PRK, RuBP, |
− | cannot be | + | 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 | + | 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"> | ||
We tested PRK in different strains. We first cloned <i>prk</i> into pSB1C3 and | We tested PRK in different strains. We first cloned <i>prk</i> into pSB1C3 and | ||
− | transformed into BL21(DE3). After 12 hours, the strain without plasmid could | + | transformed into BL21 (DE3). After 12 hours, the strain without plasmid could |
grow up to 1.4 O.D.600 in altered M9 xylose medium. The strain that contains | grow up to 1.4 O.D.600 in altered M9 xylose medium. The strain that contains | ||
PRK can grow up to 0.75 O.D.600 in normal M9 medium either. In contrast, the | PRK can grow up to 0.75 O.D.600 in normal M9 medium either. In contrast, the | ||
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<p class="pcontent"> | <p class="pcontent"> | ||
− | Fig 3. The result of PRK test in BL21(DE3). The PRK expressing strain is | + | Fig 3. The result of PRK test in BL21 (DE3). The PRK expressing strain is |
incubated | incubated | ||
in both normal M9 medium and altered M9 xylose medium to compare with the | in both normal M9 medium and altered M9 xylose medium to compare with the | ||
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<p class="pcontent"> | <p class="pcontent"> | ||
− | Fig 4. Compares the growth in M9 xylose medium of | + | Fig 4. Compares the growth in M9 xylose medium of PRK expressing strain which prk is cloned into |
high | high | ||
− | and low copy number | + | and low copy number plasmids respectively. The low copy number plasmid, pSB3K3, shows a |
− | little bit of growth retard compare to | + | little bit of growth retard compare to the control strain. However, its |
− | toxicity is much less than high copy number | + | 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 | + | BL21 (DE3), but the difference between experiement and control group is not obvious . 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 | + | 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=" | + | <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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<ol> | <ol> | ||
− | <li>Construct the ca and transform it into BL21(DE3)</li> | + | <li>Construct the <i>ca</i> and transform it into BL21 (DE3)</li> |
<li>Run the SDS-PAGE to confirm its expression</li> | <li>Run the SDS-PAGE to confirm its expression</li> | ||
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style="color:#28ff28;">BBa_K2762008</a>) into pSB1C3 plasmid after the gene | style="color:#28ff28;">BBa_K2762008</a>) into pSB1C3 plasmid after the gene | ||
is amplified | is amplified | ||
− | with PCR. We transform the plasmid into DH5 alpha and BL21(DE3). Next, we | + | with PCR. We transform the plasmid into DH5 alpha and BL21 (DE3). Next, we |
confirm its protein expression with SDS-PAGE. | confirm its protein expression with SDS-PAGE. | ||
</p> | </p> | ||
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<p class="pcontent"> | <p class="pcontent"> | ||
− | Fig 6. Confirmation of ca digestion</br> | + | Fig 6. Confirmation of <i>ca</i> digestion</br> |
− | Fig 7. Confirmation of CA expression in BL21(DE3). The expected protein size is | + | Fig 7. Confirmation of CA expression in BL21 (DE3). The expected protein size is |
27.9kDa. | 27.9kDa. | ||
</p></br> | </p></br> | ||
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We then ran the activity test of CA. In our bypass pathway, the function of CA | We then ran the activity test of CA. In our bypass pathway, the function of CA | ||
is to | is to | ||
− | convert proton and bicarbonate into water and carbon dioxide. CA activity was | + | convert proton and bicarbonate into water and carbon dioxide. CA activity was determined using the Wilbur-Anderson assay. Briefly, 9 mL ice-cold Tris−HCl (20 |
− | + | ||
mM, pH8.3) buffer and 0.2 mL enzyme were mixed and transferred to a 20 mL | mM, pH8.3) buffer and 0.2 mL enzyme were mixed and transferred to a 20 mL | ||
sample bottle, with further incubation at 0 °C with stirring. Then, 6 mL of | sample bottle, with further incubation at 0 °C with stirring. Then, 6 mL of | ||
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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 | ||
Line 248: | Line 248: | ||
<p class="pcontent"> | <p class="pcontent"> | ||
− | Fig 8. Confirmation of rbcX and rbcS digestion | + | Fig 8. Confirmation of <i>rbcX</i> and <i>rbcS</i> digestion |
− | Fig 9. Confirmation of | + | 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. | + | size is 15.3 kDA and 13.8 kDA respectively. |
</p> | </p> | ||
Line 257: | Line 257: | ||
<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. | + | 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"> | ||
− | |||
<h3>Total solution</h3> | <h3>Total solution</h3> | ||
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<li>Check the growth and carbon fixation enhancement of CA enzyme</li> | <li>Check the growth and carbon fixation enhancement of CA enzyme</li> | ||
− | <li>Compare the carbon fixation rate of W3110 and BL21(DE3) <i>E. coli</i> | + | <li>Compare the carbon fixation rate of W3110 and BL21 (DE3) <i>E. coli</i> |
strains</li> | strains</li> | ||
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<div class="row"> | <div class="row"> | ||
<a class="btn col-md-12" data-toggle="collapse" href="#Total_solution_overview" role="button" aria-expanded="false" aria-controls="multiCollapseExample1"> | <a class="btn col-md-12" data-toggle="collapse" href="#Total_solution_overview" role="button" aria-expanded="false" aria-controls="multiCollapseExample1"> | ||
− | + | Principle and Mechanism of Xylose Utilzation Index | |
<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> | ||
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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 |
− | + | ||
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 | + | that all the enzymes in our design is necessary for carbon fixation. |
</p></br> | </p></br> | ||
<p class="pcontent"> | <p class="pcontent"> | ||
− | The | + | 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 | + | 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 | + | strain that has the highest capacity of carbon fixation. |
</p></br> | </p></br> | ||
<p class="pcontent"> | <p class="pcontent"> | ||
− | To define the XUI | + | To define the XUI, we firstly made two assumptions: |
</p> | </p> | ||
<ol> | <ol> | ||
− | <li>O.D. 600 of the sample has a linear relationship to dry cell weight | + | <li>O.D. 600 of the sample has a linear relationship to dry cell weight |
(biomass). Optical density is frequently used as a means of describing the | (biomass). Optical density is frequently used as a means of describing the | ||
cell density in the broth. We measured the dry cell weight of samples in | cell density in the broth. We measured the dry cell weight of samples in | ||
different O.D. value and discovered that it has a linear relationship. We | different O.D. value and discovered that it has a linear relationship. We | ||
conclude that we can utilize O.D. value to estimate the dry cell weight. 1 | conclude that we can utilize O.D. value to estimate the dry cell weight. 1 | ||
− | 0.D. of BL21(DE3) strain per litter yields the dry cell weight of 0.8 gram.</li> | + | 0.D. of BL21 (DE3) strain per litter yields the dry cell weight of 0.8 gram.</li> |
</ol> | </ol> | ||
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<p class="pcontent"> | <p class="pcontent"> | ||
− | Fig 12. shows the dry cell weight of BL21(DE3) incubated in altered M9 xylose | + | Fig 12. shows the dry cell weight of BL21 (DE3) incubated in altered M9 xylose |
medium. | medium. | ||
A linear relationship between O.D. and dry cell weight is observed. | A linear relationship between O.D. and dry cell weight is observed. | ||
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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 | + | 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 | + | 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 | ||
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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 | + | 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"> | ||
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Before measuring the XUI, we observe the growth curve of each strain. We found | Before measuring the XUI, we observe the growth curve of each strain. We found | ||
out | out | ||
− | that W3110(L5T7) constructed strain cannot grow in altered M9 solution. | + | that W3110 (L5T7) constructed strain cannot grow in altered M9 solution. |
− | W3110(L5T7) | + | W3110 (L5T7) |
is a newly constructed strain, we are not quite certain its characteristic. We | is a newly constructed strain, we are not quite certain its characteristic. We | ||
− | eliminate this strain from the following experiment. BL21(DE3) and W3110 | + | eliminate this strain from the following experiment. BL21 (DE3) and W3110 |
constructed strains show little growth after 24 hours. | constructed strains show little growth after 24 hours. | ||
</p> | </p> | ||
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<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. | ||
</p></br> | </p></br> | ||
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<p class="pcontent"> | <p class="pcontent"> | ||
We then utilized XUI to evaluate the function of each enzyme in the pathway. We | We then utilized XUI to evaluate the function of each enzyme in the pathway. We | ||
− | first check the function of Rubisco in BL21(DE3) strain. Rubisco enzyme with | + | first check the function of Rubisco in BL21 (DE3) strain. Rubisco enzyme with |
promoter P<sub>T7</sub> (<a href="http://parts.igem.org/Part:BBa_K2762011" | promoter P<sub>T7</sub> (<a href="http://parts.igem.org/Part:BBa_K2762011" | ||
style="color:#28ff28;">BBa_K2762011</a>) was cloned into pSB1C3 and PRK | style="color:#28ff28;">BBa_K2762011</a>) was cloned into pSB1C3 and PRK | ||
with promoter P<sub>LacI</sub> (<a href="http://parts.igem.org/Part:BBa_K2762007" | with promoter P<sub>LacI</sub> (<a href="http://parts.igem.org/Part:BBa_K2762007" | ||
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 |
− | + | 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 | |
− | than the experiment and | + | 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 | |
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 | + | XUI, symbolizing that Rubisco is essential in the carbon fixation pathway. |
</p> | </p> | ||
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<p class="pcontent"> | <p class="pcontent"> | ||
− | Fig 15. Shows the growth and XUI measured in 5% CO<sub>2</sub> incubation | + | Fig 15. Shows the growth and XUI measured in 5% CO<sub>2</sub> incubation for 12 |
hours | hours | ||
− | respectively. | + | 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 470: | Line 468: | ||
style="color:#28ff28;">BBa_K2762011</a>) into pSB1C3 and | style="color:#28ff28;">BBa_K2762011</a>) into pSB1C3 and | ||
cloned PRK with P<sub>LacI</sub> promoter and CA with P<sub>T7</sub> | cloned PRK with P<sub>LacI</sub> promoter and CA with P<sub>T7</sub> | ||
− | promoter(<a href="http://parts.igem.org/Part:BBa_K2762013" style="color:#28ff28;">BBa_K2762013</a>) | + | promoter (<a href="http://parts.igem.org/Part:BBa_K2762013" style="color:#28ff28;">BBa_K2762013</a>) |
− | into pSB3K3. Two plasmids are then co-transformed into BL21(DE3). We measured | + | into pSB3K3. Two plasmids are then co-transformed into BL21 (DE3). We measured |
the | the | ||
XUI of this strain and compare with the previous strain that only contains PRK | XUI of this strain and compare with the previous strain that only contains PRK | ||
Line 496: | Line 494: | ||
</p></br> | </p></br> | ||
− | <h8>XUI Comparison between BL21(DE3) and W3110</h8></br></br> | + | <h8>XUI Comparison between BL21 (DE3) and W3110</h8></br></br> |
<p class="pcontent"> | <p class="pcontent"> | ||
− | We then compare the XUI value between BL21(DE3) and W3110 constructed strain. | + | We then compare the XUI value between BL21 (DE3) and W3110 constructed strain. |
When we design our IDT sequence, we link the CA directly to the promoter P<sub>LacI</sub>, | When we design our IDT sequence, we link the CA directly to the promoter P<sub>LacI</sub>, | ||
so we could not transform CA construct into W3110 strain. We thus compare the | so we could not transform CA construct into W3110 strain. We thus compare the | ||
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 | + | 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 517: | Line 514: | ||
<ol> | <ol> | ||
− | <li>W3110 “wildtype” strain has more flexible metabolic network | + | <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> | ||
</ol> | </ol> | ||
Line 531: | Line 528: | ||
<p class="pcontent"> | <p class="pcontent"> | ||
− | Fig 17. Shows the growth and the XUI of BL21(DE3) and W3110 strains. | + | Fig 17. Shows the growth and the XUI of BL21 (DE3) and W3110 strains. |
</p></br> | </p></br> | ||
<p class="pcontent"> | <p class="pcontent"> | ||
− | We finally concluded that the efficiency of the bypass pathway in BL21(DE3) is | + | We finally concluded that the efficiency of the bypass pathway in BL21 (DE3) is |
better than that in the W3110 strain. | better than that in the W3110 strain. | ||
</p></br> | </p></br> | ||
Line 543: | 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 560: | Line 557: | ||
<p class="pcontent"> | <p class="pcontent"> | ||
− | 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 | + | 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 575: | Line 572: | ||
<div id="Carbon_fixation"> | <div id="Carbon_fixation"> | ||
− | </ | + | <h3>Carbon fixation</h3> |
− | + | <div class="row"> | |
− | <div id=" | + | <a class="btn col-md-12" data-toggle="collapse" href="#Estimation_Carbon_fixation" role="button" aria-expanded="false" aria-controls="multiCollapseExample1"> |
− | <p class="pcontent"> | + | Amount of Carbon Fixed: 0.575 mg / l * hr |
− | + | <i class="fa fa-arrow-down fa-10" aria-hidden="true"></i> | |
− | + | </a> | |
− | + | </div> | |
− | + | <div class="collapse multi-collapse" id="Estimation_Carbon_fixation"> | |
− | + | <div class="card card-body"> | |
− | + | <div id="pt"> | |
+ | <p class="pcontent"> | ||
+ | To find out how much and how efficient genetically engineered <i>E. coli</i> | ||
+ | can fix | ||
+ | carbon dioxide, we use the material balance concept to evaluate the | ||
+ | heterotrophic CO<sub>2</sub> fixation process. Consider a system composed of a | ||
+ | single | ||
+ | component, the general material balance can be written as: | ||
− | + | $${\{Input\ to\ the\ system\}\ –\ \{Output\ to\ the\ system\}\ =\ | |
− | + | \{Accumulation\ in\ the\ system\}}$$ | |
− | + | A system can be defined as an arbitrary portion of a process considered for | |
− | + | analysis, in which in this case, is an engineered carbon capturing <i>E. coli</i>. | |
− | + | </p> | |
− | + | </div> | |
− | + | <img class="contentimg" src=""> | |
− | + | <div id="pt"> | |
− | + | <p class="pcontent"> | |
− | + | 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 | |
− | + | contains | |
− | + | 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 | |
− | + | may neither be created nor destroyed, the material balance for carbon in an | |
− | + | engineered <i>E. coli</i> may simply be written as | |
− | + | $${\{C_{CO_2}\ in\}\ +\ \{C_{xylose}\}\ -\ \{C_{CO_2}\ out\}\ -\ \{C_{waste}\}\ | |
− | + | =\ \{C_{biomass}\}...(1)}$$ | |
− | + | Considering the difficulties in measuring carbon in <i>E. coli</i> metabolic | |
− | + | waste and | |
− | + | that C<sub>waste</sub> would be positive, the equation reduces to | |
− | + | $${\{C_{CO_2}\ in\}\ -\ \{C_{CO_2}\ out\}\ ≥\ \{C_{biomass}\}\ -\ | |
− | + | \{C_{xylose}\}...(2)}$$ | |
− | + | Let {C<sub>CO<sub>2</sub></sub> net}= {C<sub>CO<sub>2</sub></sub> in} - {C<sub>CO<sub>2</sub></sub> | |
− | + | out}, equation (2) further simplifies to | |
− | + | $${\{C_{CO_2}\ net\}\ ≥\ \{C_{biomass}\}\ -\ \{C_{xylose}\}...(3)}$$ | |
− | + | If C<sub>waste</sub> is very small and negligible, we can obtain the net amount | |
− | + | of carbon | |
− | + | dioxide fixed over time. If, on the contrary, C<sub>waste</sub> cannot be | |
− | + | neglected, | |
− | + | equation (3) allows us to estimate the minimum net amount of carbon dioxide | |
− | + | fixed. | |
− | + | </p> | |
− | + | <p class="pcontent"> | |
− | + | C<sub>biomass</sub> can be calculate by multiplying O.D. 600 to DCW and mass | |
− | + | percent of carbon in <i>E. coli</i> biomass. The O.D. 600 of engineered <i>E. | |
− | + | coli</i> is | |
− | + | measured after a 12-hour cultivation and the result obtained is 0.45O.D. . Yin | |
− | + | Li et al. reported that dry cell weight (DCW) of <i>E. coli</i> is | |
− | + | $${0.35g\over L ∙ 𝑂.𝐷. 600}$$ | |
− | + | , determined by experiment. <i>E. coli</i> biomass contains 48% of carbon by | |
− | + | mass. | |
− | + | $${C_{biomass}\ =\ 0.4511\ ×\ 0.35\ ×\ 48\%}$$ | |
− | + | $${=\ 0.0758\ g/L}$$ | |
− | + | </p> | |
− | + | <p class="pcontent"> | |
− | + | On the other hand, C<sub>xylose</sub> can be calculated by multiplying the | |
− | + | amount of | |
− | + | 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 | |
− | + | concentration of reducing sugar and the result obtained is 0.1723g of xylose | |
− | + | consumed per liter of M9 medium. Carbon mass percentage of xylose | |
− | + | is 40%. | |
− | + | $${C_{xylose}\ =\ 0.1723\ ×\ 40\%\ =\ 0.0689\ g/L}$$ | |
− | + | By equation (3) | |
− | + | $${C_{CO_2\ net}\ =\ 0.0758\ -\ 0.0689}$$ | |
− | + | $${=\ 0.0069\ g/L}$$ | |
− | + | Since the <i>E. coli</i> has been cultured for 12 hours, we can calculate the | |
− | + | rate of | |
− | + | carbon fixation by | |
− | + | $${Rate\ of\ carbon\ fixation\ =\ {𝐶_{𝐶𝑂_2\ 𝑛𝑒𝑡}\over 12}}$$ | |
− | + | $${=\ {0.0069\over 12}}$$ | |
− | + | $${=\ 0.575\ {mg\over L ∙hr}}$$ | |
− | + | 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: | |
− | + | ||
− | + | ||
− | + | ||
− | |||
− | |||
− | |||
</p> | </p> | ||
− | + | <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> | ||
+ | </div> | ||
+ | </div> | ||
</div> | </div> | ||
− | |||
− | |||
</div> | </div> | ||
− | |||
<div id="pH_Senesing"> | <div id="pH_Senesing"> | ||
− | |||
<h3>pH sensing system</h3> | <h3>pH sensing system</h3> | ||
Line 700: | Line 700: | ||
<li>Construct the pH sensing system</li> | <li>Construct the pH sensing system</li> | ||
− | <li>Measure and characterize the short-term fluorescence intensity of P<sub>asr</sub></li> | + | <li>Measure and characterize the short-term fluorescence intensity of P<sub>asr</sub> (<a href="http://parts.igem.org/Part:BBa_K1231000" |
+ | style="color:#28ff28;">BBa_K1231000</a>)</li> | ||
+ | |||
+ | <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> | ||
+ | <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 714: | 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 | + | 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 | ||
− | then cloned into pSB1C3 plasmid and transformed into BL21(DE3). | + | then cloned into pSB1C3 plasmid and transformed into BL21 (DE3). |
</p> | </p> | ||
</div> | </div> | ||
Line 726: | 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 734: | 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 | + | 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 748: | Line 754: | ||
<p class="pcontent"> | <p class="pcontent"> | ||
− | Based on the data has shown above, we could conclude that | + | 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 | + | 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 759: | 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 | + | 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 | ||
− | + | weak acidic environment. | |
</p> | </p> | ||
− | <img class="contentimg fig3" src="https://static.igem.org/mediawiki/2018/a/ | + | <img class="contentimg fig3" src="https://static.igem.org/mediawiki/2018/a/a3/T--NCKU_Tainan--wifi_pgada_flrescent.png"> |
<p class="pcontent"> | <p class="pcontent"> | ||
− | Fig | + | Fig 20. The data shows the fluorescence intensity (absorbance: 485 nm, |
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 780: | 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>. 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 Improvement page. | + | the <a href="https://2018.igem.org/Team:NCKU_Tainan/Improve" |
+ | style="color:#28ff28;">Improvement</a> page. | ||
</p> | </p> | ||
− | + | </div> | |
− | < | + | |
+ | <div id="References"> | ||
<h3>References</h3> | <h3>References</h3> | ||
Line 806: | Line 825: | ||
</div> | </div> | ||
− | + | ||
</div> | </div> | ||
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$(document).ready(function () { | $(document).ready(function () { | ||
$(window).scroll(function () { | $(window).scroll(function () { | ||
− | if ($(this).scrollTop() >= | + | if ($(this).scrollTop() >= 500) { |
var position = $("#sidelist").position(); | var position = $("#sidelist").position(); | ||
if (position == undefined) {} else { | if (position == undefined) {} else { |
Latest revision as of 03:26, 18 October 2018
Results
Hard Work Pays Off