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− | <link rel="stylesheet" href="https://2018.igem.org/Template:NCKU_Tainan/css/ | + | <link rel="stylesheet" href="https://2018.igem.org/Template:NCKU_Tainan/css/results?action=raw&ctype=text/css"> |
</head> | </head> | ||
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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="#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> | ||
Line 37: | Line 41: | ||
each strain.</li> | each strain.</li> | ||
<li>Estimate the carbon fixation amount with our experiment result.</li> | <li>Estimate the carbon fixation amount with our experiment result.</li> | ||
− | <li> | + | <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_K1962013</a>).</li> | ||
</ol> | </ol> | ||
</div> | </div> | ||
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<div id="Construction"> | <div id="Construction"> | ||
− | + | <h3>Construction and functional test </h3></br> | |
− | <h3>Construction and functional test </h3> | + | |
<div id="pt"> | <div id="pt"> | ||
− | <h8>PRK</h8></br> | + | <h8>PRK (PHOSPHORIBULOKINASE)</h8></br></br> |
<p class="pcontent"> | <p class="pcontent"> | ||
Achievements:</br> | Achievements:</br> | ||
Line 61: | 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 | + | 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 | |
+ | 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> | ||
</div> | </div> | ||
− | <img class="contentimg" src=" "> | + | <img class="contentimg" src="https://static.igem.org/mediawiki/2018/8/80/T--NCKU_Tainan--Results_fig_1.jpg"> |
<div id="pt"> | <div id="pt"> | ||
<p class="pcontent"> | <p class="pcontent"> | ||
− | Fig | + | Fig 1. Confirmation of <i>prk</i> digestion.</br> |
− | Fig | + | Fig 2. Confirmation of PRK expression in DH5 alpha. The expected protein size is |
37.7kDa. | 37.7kDa. | ||
</p> | </p> | ||
Line 86: | Line 93: | ||
We initially decided to test its function by HPLC to measure the amount of RuBP | We initially decided to test its function by HPLC to measure the amount of RuBP | ||
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 | ||
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 | ||
Line 99: | Line 107: | ||
<p class="pcontent"> | <p class="pcontent"> | ||
− | We tested PRK in different strains. We first cloned prk 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 | + | PRK can grow up to 0.75 O.D.600 in normal M9 medium either. In contrast, the |
PRK strain that grew in altered M9 xylose medium showed no growth at all. The | PRK strain that grew in altered M9 xylose medium showed no growth at all. The | ||
result shows that PRK can suppress/inhibit the growth, which matches to our | result shows that PRK can suppress/inhibit the growth, which matches to our | ||
Line 108: | Line 116: | ||
</p> | </p> | ||
− | <img class="contentimg" src=" "> | + | <img class="contentimg fig3" src="https://static.igem.org/mediawiki/2018/3/3d/T--NCKU_Tainan--Results_fig_3.png"> |
<p class="pcontent"> | <p class="pcontent"> | ||
− | Fig | + | 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 | ||
Line 121: | Line 129: | ||
<p class="pcontent"> | <p class="pcontent"> | ||
− | Although the function of PRK | + | Although the function of PRK has been confirmed, we would like to lower the |
expression of it to minimize the growth arrest. We thus cloned the part into | expression of it to minimize the growth arrest. We thus cloned the part into | ||
pSB3K3, a low copy number plasmid to lower its protein expression. We then | pSB3K3, a low copy number plasmid to lower its protein expression. We then | ||
Line 128: | Line 136: | ||
shows a | shows a | ||
little growth arrest comparing to the strain without plasmid. The growth of it | little growth arrest comparing to the strain without plasmid. The growth of it | ||
− | + | exceeds that of PRK expressed in pSB1C3. We can regulate the expression of PRK | |
via | via | ||
high or low copy number plasmid to optimize the growth and carbon fixation | high or low copy number plasmid to optimize the growth and carbon fixation | ||
Line 134: | Line 142: | ||
</p> | </p> | ||
− | <img class="contentimg" src=" "> | + | <img class="contentimg fig4" src="https://static.igem.org/mediawiki/2018/3/3a/T--NCKU_Tainan--Results_fig_4.PNG"> |
<p class="pcontent"> | <p class="pcontent"> | ||
− | Fig. | + | 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> | ||
<p class="pcontent"> | <p class="pcontent"> | ||
− | We also transformed pSB3K3-prk 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 protein may be lower than that of high copy number plasmid. The | + | expression of the protein may be lower than that of high copy number plasmid. |
− | pressure tolerance of W3110 strain may also lessen the | + | The |
+ | pressure tolerance of W3110 strain may also lessen the influence of toxicity by | ||
PRK. | PRK. | ||
</p> | </p> | ||
− | <img class="contentimg" src=" "> | + | <img class="contentimg fig5" src="https://static.igem.org/mediawiki/2018/f/f6/T--NCKU_Tainan--Results_fig_4_2.PNG"> |
+ | <p class="pcenter"> | ||
+ | Fig 5. The result of PRK test in W3110 | ||
+ | </p></br> | ||
</div> | </div> | ||
<div id="pt"> | <div id="pt"> | ||
− | <h8>CA</h8></br> | + | <h8>CA (Carbonic Anhydrase)</h8></br></br> |
<p class="pcontent"> | <p class="pcontent"> | ||
Achievements:</br> | Achievements:</br> | ||
Line 166: | Line 177: | ||
<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> | ||
Line 174: | Line 185: | ||
<p class="pcontent"> | <p class="pcontent"> | ||
− | We cloned the DNA fragments into pSB1C3 plasmid after the gene is amplified | + | We cloned the DNA fragments (<a href="http://parts.igem.org/Part:BBa_K2762008" |
− | with PCR. We transform the plasmid into DH5 | + | style="color:#28ff28;">BBa_K2762008</a>) into pSB1C3 plasmid after the gene |
+ | is amplified | ||
+ | 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> | ||
− | <img class="contentimg" src=" "> | + | <img class="contentimg" src="https://static.igem.org/mediawiki/2018/0/04/T--NCKU_Tainan--Results_fig_5.jpg"> |
<p class="pcontent"> | <p class="pcontent"> | ||
− | Fig. | + | Fig 6. Confirmation of <i>ca</i> digestion</br> |
− | Fig. | + | Fig 7. Confirmation of CA expression in BL21 (DE3). The expected protein size is |
27.9kDa. | 27.9kDa. | ||
</p></br> | </p></br> | ||
Line 196: | Line 209: | ||
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. | + | 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 | |
− | + | sample bottle, with further incubation at 0 °C with stirring. Then, 6 mL of | |
− | the | + | ice-cold CO<sub>2</sub>-saturated solution was added immediately into the sample bottle |
− | + | 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 | |
− | the | + | 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 | ||
+ | CA, respectively. The enzyme activity of our CA is 21.8 | ||
unit/liter. | unit/liter. | ||
To confirm the contribution of the CA to the whole pathway, we also ran the | To confirm the contribution of the CA to the whole pathway, we also ran the | ||
Line 211: | Line 226: | ||
<div id="pt"> | <div id="pt"> | ||
− | <h8>Rubisco</h8></br> | + | <h8>Rubisco</h8></br></br> |
<p class="pcontent"> | <p class="pcontent"> | ||
Achievements:</br> | Achievements:</br> | ||
Line 219: | Line 234: | ||
<li>Construction and digestion of DNA gel shows that the size of it was right</li> | <li>Construction and digestion of DNA gel shows that the size of it was right</li> | ||
− | <li>The SDS-PAGE of | + | <li>The SDS-PAGE of Rubisco showed that the expression of Rubisco in the expected size</li> |
</ol></br> | </ol></br> | ||
<p class="pcontent"> | <p class="pcontent"> | ||
− | We constructed | + | We constructed rubisco fragments from IDT DNA synthesis. After PCR amplification of |
the three subunits, rubisco is then cloned into pSB1C3 and transformed into | the three subunits, rubisco 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: | are shown below: | ||
</p> | </p> | ||
− | <img class="contentimg" src=" "> | + | |
+ | <img class="contentimg" src="https://static.igem.org/mediawiki/2018/c/cb/T--NCKU_Tainan--Results_fig_9.jpg"> | ||
<p class="pcontent"> | <p class="pcontent"> | ||
− | Fig. | + | Fig 8. Confirmation of <i>rbcX</i> and <i>rbcS</i> digestion |
− | Fig. | + | Fig 9. Confirmation of <i>rbcX</i> and <i>rbcS</i> expression in BL21 (DE3). The expected protein |
− | + | size is 15.3 kDA and 13.8 kDA respectively. | |
</p> | </p> | ||
+ | <img class="contentimg" src="https://static.igem.org/mediawiki/2018/0/0d/T--NCKU_Tainan--Results_fig_7.jpg"> | ||
+ | |||
+ | <p class="pcontent"> | ||
+ | Fig 10. Confirmation of <i>rbcL</i> digestion.</br> | ||
+ | |||
+ | Fig 11. Confirmation of <i>rbcL</i> expression in DH5 alpha. The expected protein size is | ||
+ | 52.37 kDa.</br> | ||
+ | </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 | ||
− | to determine the activity of | + | to determine the activity of Rubisco by thin-layer chromatographic has been |
reported. However, due to time concern, we are not capable of measuring the | reported. However, due to time concern, we are not capable of measuring the | ||
enzyme activity of Rubisco with this method. We finally confirm its function | enzyme activity of Rubisco with this method. We finally confirm its function | ||
Line 252: | Line 277: | ||
<div id="Total_solution"> | <div id="Total_solution"> | ||
− | |||
<h3>Total solution</h3> | <h3>Total solution</h3> | ||
<div id="pt"> | <div id="pt"> | ||
− | <h8>Rubisco</h8></br> | + | <h8>Rubisco</h8></br></br> |
<p class="pcontent"> | <p class="pcontent"> | ||
Achievements:</br> | Achievements:</br> | ||
Line 269: | Line 293: | ||
<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) E. coli strains</li> | + | <li>Compare the carbon fixation rate of W3110 and BL21 (DE3) <i>E. coli</i> |
+ | strains</li> | ||
− | <li>Compare different | + | <li>Compare different CO<sub>2</sub> incubation environment</li> |
</ol></br> | </ol></br> | ||
− | < | + | <div class="row"> |
− | <p class="pcontent"> | + | <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> | |
− | + | </a> | |
− | + | </div> | |
− | + | <div class="collapse multi-collapse" id="Total_solution_overview"> | |
− | + | <div class="card card-body"> | |
− | + | <p class="pcontent"> | |
− | + | In the total solution experiment, we strive to measure the carbon fixation | |
− | + | amount of each sample. After reading numerous publications, we found out that | |
− | + | previous researches determine the efficiency of carbon fixation via measuring | |
− | + | the decrease of carbon dioxide concentration in the closed system or measure | |
− | + | 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 | |
− | + | decrease of carbon dioxide concentration in the closed system is also | |
+ | impractical for us since we have too much test samples. A new method to measure | ||
+ | multiple samples in the short period of time is developed by our team. We are | ||
+ | able to evaluate the fixation efficiency of each sample with the optical | ||
+ | density | ||
+ | O.D. 600 and xylose consumption. We have measure various construction to prove | ||
+ | that all the enzymes in our design is necessary for carbon fixation. | ||
+ | </p></br> | ||
− | + | <p class="pcontent"> | |
− | + | 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 the trace elements. | |
− | + | Since the concentration of LB medium is too low, it doesn’t contribute the | |
− | + | carbon source of the bacteria. | |
− | + | </p></br> | |
− | + | <p class="pcontent"> | |
− | + | 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 | |
− | + | strain that has the highest capacity of carbon fixation. | |
− | + | </p></br> | |
− | + | <p class="pcontent"> | |
− | + | To define the XUI, we firstly made two assumptions: | |
− | + | </p> | |
− | + | <ol> | |
− | + | <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 | |
− | + | 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 | |
− | + | 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> | |
− | + | </ol> | |
− | + | <img class="contentimg fig10" src="https://static.igem.org/mediawiki/2018/6/64/T--NCKU_Tainan--Results_fig_10.png"> | |
− | + | <p class="pcontent"> | |
− | + | Fig 12. shows the dry cell weight of BL21 (DE3) incubated in altered M9 xylose | |
− | + | medium. | |
− | + | A linear relationship between O.D. and dry cell weight is observed. | |
− | + | </p></br> | |
− | + | <ol start="2"> | |
− | + | <li>The elemental formula of <i>E. coli</i> should be fixed or varies within a | |
− | + | small range. Although the formula may have variations in different | |
− | + | growth condition, we assume that such error can be ignored during the | |
− | + | following calculation.</li> | |
− | + | </ol></br> | |
− | + | <p class="pcontent"> | |
− | + | After these two assumptions, the Xylose Utilization Index is designed to | |
− | + | evaluate | |
− | + | the carbon fixation ability of each strain. The definition of the index is | |
− | + | xylose | |
− | + | consumption over O.D. 600. O.D. 600 measurement can be viewed as the weight of | |
− | + | carbon of the bacteria. The index shows the ratio of xylose consumption per | |
− | + | biomass. For wild-type <i>E. coli</i>, it only consumes xylose (the sole carbon | |
− | + | source | |
− | + | provided in our medium) as its carbon source. Although some native <i>E. coli</i> | |
− | + | pathway | |
− | + | may utilize CO<sub>2</sub> (such as lipid synthesis), the amount is too small | |
− | + | to consider. | |
− | + | As | |
− | + | for engineered strain, carbon dioxide can be utilized as it’s carbon source. By | |
− | + | producing the same amount of carbon biomass, it requires less xylose. We can | |
− | + | thus | |
− | + | compare the XUI of each strain to determine the strain that fixes | |
+ | carbon. | ||
+ | The less the XUI in the sample, the more possibility that it fixes carbon. | ||
+ | </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"> | ||
− | + | <p class="pcontent"> | |
+ | We use the Dinitrosalicylic Acid (DNS) reducing sugar assay to measure the | ||
+ | xylose | ||
+ | concentration in the medium. Under base solution, DNS will turn to brown color | ||
+ | while reacting with reductive sugar in high temperature. In the specific | ||
+ | temperature range, the color will have a linear relationship with the reductive | ||
+ | sugar | ||
+ | concentration. We can thus measure the xylose concentration at O.D.540. | ||
+ | </p> | ||
− | + | <img class="contentimg fig11" src="https://static.igem.org/mediawiki/2018/3/3f/T--NCKU_Tainan--Results_Fig_10.PNG"> | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | <p class="pcontent"> | |
+ | Fig 13. Shows the calibration line of DNS assay kit. | ||
+ | </p></br> | ||
− | + | <p class="pcontent"> | |
− | + | Before measuring the XUI, we observe the growth curve of each strain. We found | |
− | + | out | |
− | + | that W3110 (L5T7) constructed strain cannot grow in altered M9 solution. | |
− | + | W3110 (L5T7) | |
− | + | is a newly constructed strain, we are not quite certain its characteristic. We | |
− | + | eliminate this strain from the following experiment. BL21 (DE3) and W3110 | |
− | + | constructed strains show little growth after 24 hours. | |
− | + | </p> | |
− | + | <img class="contentimg fig12" src="https://static.igem.org/mediawiki/2018/5/51/T--NCKU_Tainan--Results_Fig_11.PNG"> | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | <h8>Total solution check: Function of Rubisco</h8> | + | <p class="pcontent"> |
+ | 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 | ||
+ | strains show growth after 24hours. | ||
+ | </p></br> | ||
+ | </div> | ||
+ | </div> | ||
+ | <br> | ||
+ | <h8>Total solution check: Function of Rubisco</h8></br></br> | ||
<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 | + | 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 | + | capable of using CO<sub>2</sub>. We found out that the strain without Rubisco |
− | XUI, symbolizing that | + | has higher |
+ | XUI, symbolizing that Rubisco is essential in the carbon fixation pathway. | ||
</p> | </p> | ||
− | <img class="contentimg col-6" src=" "> | + | <img class="contentimg col-6" src="https://static.igem.org/mediawiki/2018/3/35/T--NCKU_Tainan--Results_fig_13.png"> |
− | <img class="contentimg col-6" src=" "> | + | <img class="contentimg col-6" src="https://static.igem.org/mediawiki/2018/0/0e/T--NCKU_Tainan--Results_fig_14.png"> |
<p class="pcontent"> | <p class="pcontent"> | ||
− | Fig. | + | Fig 15. Shows the growth and XUI measured in 5% CO<sub>2</sub> incubation for 12 |
− | respectively. | + | hours |
+ | 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> | ||
</p></br> | </p></br> | ||
− | <h8>Total solution check: Function of CA</h8> | + | <h8>Total solution check: Function of CA</h8></br></br> |
<p class="pcontent"> | <p class="pcontent"> | ||
From the above results, we discovered that although Rubisco and | From the above results, we discovered that although Rubisco and | ||
− | + | PRK alone can enhance the utilization rate of carbon dioxide, the growth and | |
utilization ability didn’t meet our expectations. The third important enzyme | utilization ability didn’t meet our expectations. The third important enzyme | ||
came into play: CA enzyme. We cloned Rubisco (<a href="http://parts.igem.org/Part:BBa_K2762011" | came into play: CA enzyme. We cloned Rubisco (<a href="http://parts.igem.org/Part:BBa_K2762011" | ||
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 | ||
and Rubisco. We found out that CA can raise the growth and lower the XUI. We | and Rubisco. We found out that CA can raise the growth and lower the XUI. We | ||
− | infer that CA can enhance the intracellular | + | infer that CA can enhance the intracellular CO<sub>2</sub> concentration and |
+ | thus increase | ||
the carbon flux of the bypass pathway. The efficiency of the bypass pathway is | the carbon flux of the bypass pathway. The efficiency of the bypass pathway is | ||
thus been increased. | thus been increased. | ||
</p> | </p> | ||
− | <img class="contentimg col-6" src=" "> | + | <img class="contentimg col-6" src="https://static.igem.org/mediawiki/2018/9/99/T--NCKU_Tainan--Results_fig_15.png"> |
− | <img class="contentimg col-6" src=" "> | + | <img class="contentimg col-6 fig14_2" src="https://static.igem.org/mediawiki/2018/5/54/T--NCKU_Tainan--Results_fig_16.png"> |
<p class="pcontent"> | <p class="pcontent"> | ||
− | Fig. | + | Fig 16. Shows the growth and XUI comparison of each strain. All the tested |
− | strains are incubated in 5% | + | strains are incubated in 5% CO<sub>2</sub> incubator for 12 hr. 0.1mM of IPTG |
− | induce | + | was added to |
− | construction has been increased with the CA | + | induce protein expression. We can observe that growth speed of the |
+ | construction has been increased with the CA and the XUI of the strain that | ||
contains complete three enzymes was the lowest compared to the strain without | contains complete three enzymes was the lowest compared to the strain without | ||
plasmid or the strain that only contains PRK and Rubisco, stating that three | plasmid or the strain that only contains PRK and Rubisco, stating that three | ||
− | enzymes are required to | + | enzymes are required to optimize the carbon fixing bypass pathway. |
</p></br> | </p></br> | ||
− | <h8>XUI Comparison between BL21(DE3) and W3110</h8> | + | <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 | + | 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 | ||
XUI of strains that only contains Rubisco and PRK (<a href="http://parts.igem.org/Part:BBa_K2762011" | XUI of strains that only contains Rubisco and PRK (<a href="http://parts.igem.org/Part:BBa_K2762011" | ||
− | style="color:#28ff28;">BBa_K2762011</a>) , | + | style="color:#28ff28;">BBa_K2762011</a>), |
(<a href="http://parts.igem.org/Part:BBa_K2762007" style="color:#28ff28;">BBa_K2762007</a>). | (<a href="http://parts.igem.org/Part:BBa_K2762007" style="color:#28ff28;">BBa_K2762007</a>). | ||
− | We found out that both | + | We found out that both strains show similar trend: the XUI will |
be lower with the expression of the constructed protein. The growth condition | be lower with the expression of the constructed protein. The growth condition | ||
of both constructed strains is similar for the first 12 hours. We then compare | of both constructed strains is similar for the first 12 hours. We then compare | ||
− | the difference of XUI between two E. coli strain. We found out that both strain | + | the difference of XUI between two <i>E. coli</i> strain. We found out that both |
− | shows similar trend: the XUI will be lower with the expression of the | + | strain |
− | + | 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 476: | 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> | ||
− | <img class="contentimg col-6" src=" "> | + | <img class="contentimg col-6" src="https://static.igem.org/mediawiki/2018/0/0c/T--NCKU_Tainan--Results_fig_17.png"> |
− | <img class="contentimg col-6" src=" "> | + | <img class="contentimg col-6 fig14_2" src="https://static.igem.org/mediawiki/2018/5/52/T--NCKU_Tainan--Results_fig_18.png"> |
<p class="pcontent"> | <p class="pcontent"> | ||
− | Fig. | + | 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> | ||
− | <h8>Incubation under different | + | <h8>Incubation under different CO<sub>2</sub> concentration</h8></br></br> |
<p class="pcontent"> | <p class="pcontent"> | ||
− | Finally, we compare the XUI under different | + | Finally, we compare the XUI under different CO<sub>2</sub> concentration. We |
− | bacteria in normal incubator without | + | incubated the engineered |
− | that maintains 5% | + | bacteria in normal incubator without CO<sub>2</sub> input and the cell culture |
− | incubator has lower the XUI. The supply of sufficient | + | incubator |
− | efficiency of the bypass pathway and enhance the growth. We can | + | that maintains 5% CO<sub>2</sub> concentration. We observed that the strain in |
− | our constructed pathway can | + | 5% CO<sub>2</sub> |
+ | incubator has lower the XUI. The supply of sufficient CO<sub>2</sub> can | ||
+ | increase the | ||
+ | efficiency of the bypass pathway and enhance the growth. We can conclude that | ||
+ | our constructed pathway can utilize carbon dioxide as one of its carbon | ||
source from this result. | source from this result. | ||
</p> | </p> | ||
− | <img class="contentimg col-6" src=" "> | + | <img class="contentimg col-6" src="https://static.igem.org/mediawiki/2018/6/67/T--NCKU_Tainan--Results_Fig_15.PNG"> |
− | <img class="contentimg col-6" src=" "> | + | <img class="contentimg col-6 fig16_2" src="https://static.igem.org/mediawiki/2018/7/71/T--NCKU_Tainan--Results_fig_20.png"> |
<p class="pcontent"> | <p class="pcontent"> | ||
− | Fig. | + | Fig 18. The comparison of the growth and the XUI of the BL21 (DE3) that contains |
− | all three enzymes in normal incubator and 5% | + | all three enzymes in normal incubator and 5% CO<sub>2</sub> incubator. The |
− | + | strain grown in | |
− | strain can use | + | CO<sub>2</sub> incubator showed better growth and lower XUI, which indicates |
+ | that our | ||
+ | strain can use CO<sub>2</sub> as a carbon source in the presence of high CO<sub>2</sub> | ||
+ | level. | ||
</p> | </p> | ||
Line 527: | 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 id="pH_Senesing"> | ||
+ | <h3>pH sensing system</h3> | ||
+ | |||
+ | </br> | ||
+ | <h8>Achievements:</h8></br> | ||
+ | |||
+ | <ol> | ||
+ | |||
+ | <li>Construct the pH sensing system</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> | ||
+ | |||
+ | </br> | ||
+ | <h8>Construction of the pH sensing system</h8></br> | ||
+ | |||
+ | <div id="pt"> | ||
<p class="pcontent"> | <p class="pcontent"> | ||
− | + | We construct both promoters with PCR, using primer as templates since the size | |
− | + | of it is small. For the construction of P<sub>asr</sub>, we cloned into the | |
− | + | plasmid that | |
− | + | contains both rbs and GFP. For the construction of P<sub>gadA</sub>, we took | |
− | + | the | |
− | + | constructed part from 2016 Dundee iGEM team as a reference. Both parts were | |
− | + | then cloned into pSB1C3 plasmid and transformed into BL21 (DE3). | |
+ | </p> | ||
+ | </div> | ||
− | + | </br> | |
+ | <h8>Fluorescence intensity measurement of P<sub>asr</sub></h8></br> | ||
− | + | <div id="pt"> | |
+ | <p class="pcontent"> | ||
+ | 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 | ||
+ | to report the abnormal acidity of the medium. We thus determine to measure the | ||
+ | fluorescence intensity in a short period of time. We first incubated the | ||
+ | bacteria to log phase (within 2 hour) with LB medium. We then centrifuged the | ||
+ | broth and suspended the pellet with pH modified M9 medium (the pH value is | ||
+ | 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 | ||
+ | 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 | ||
+ | fluorescence had the peak at pH value of 4.25. | ||
+ | </p> | ||
− | + | <img class="contentimg fig3" src="https://static.igem.org/mediawiki/2018/4/44/T--NCKU_Tainan--Results_fig_21.png"> | |
− | + | <p class="pcontent"> | |
+ | Fig 19. The data shows the fluorescence intensity (absorbance: 485 nm, | ||
+ | excitation: | ||
+ | 535 nm) expressed by P<sub>asr</sub> in different pH. | ||
+ | </p></br> | ||
− | + | <p class="pcontent"> | |
− | + | 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 | ||
+ | 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 | ||
+ | that this system can also be applied to various bio-detection system. | ||
+ | </p> | ||
− | + | </br> | |
+ | <h8>Fluorescence intensity measurement of P<sub>gadA</sub></h8></br></br> | ||
− | + | <p class="pcontent"> | |
+ | 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 | ||
+ | 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 | ||
+ | modified with 1M HCl). The induction of P<sub>gadA</sub> is observed under | ||
+ | neutral and | ||
+ | weak acidic environment. | ||
+ | </p> | ||
− | + | <img class="contentimg fig3" src="https://static.igem.org/mediawiki/2018/a/a3/T--NCKU_Tainan--wifi_pgada_flrescent.png"> | |
− | + | <p class="pcontent"> | |
− | the | + | Fig 20. The data shows the fluorescence intensity (absorbance: 485 nm, |
− | + | excitation: 535 nm) expressed by P<sub>gadA</sub> in different pH. | |
+ | </p> | ||
+ | <br> | ||
+ | <h8>Improvement of P<sub>gadA</sub></h8></br></br> | ||
− | + | <p class="pcontent"> | |
+ | We found out that the fluorescence intensity of P<sub>gadA</sub> is much lower | ||
+ | than the | ||
+ | P<sub>asr</sub> and would like to improve the sensitivity of this biobrick. We | ||
+ | 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 | ||
+ | the <a href="https://2018.igem.org/Team:NCKU_Tainan/Improve" | ||
+ | style="color:#28ff28;">Improvement</a> page. | ||
</p> | </p> | ||
</div> | </div> | ||
+ | |||
+ | <div id="References"> | ||
+ | <h3>References</h3> | ||
+ | <ol> | ||
+ | <li class="smallp">F. Gong, G. Liu, X. Zhai, J. Zhou, Z. Cai, Y. Li, | ||
+ | Quantitative analysis of an engineered CO<sub>2</sub>-fixing Escherichia Coli | ||
+ | reveals great potential of heterotrophic CO<sub>2</sub> fixation. Biotechnology for | ||
+ | Biofuels,8(1). doi:10.1186/s13068-015-0268-1 </li> | ||
+ | <li class="smallp">U. V. Stockar, J. Liu, Does microbial life always | ||
+ | feed on negative entropy? Thermodynamic analysis of microbial growth. | ||
+ | Biochimica Et Biophysica Acta (BBA) - Bioenergetics,1412(3), 191-211. | ||
+ | doi:10.1016/s0005-2728(99)00065-1 </li> | ||
+ | <li class="smallp">2016 Dundee iGEM team</li> | ||
+ | <li class="smallp"> S. Chakrabarti, S. Bhattacharya, S. K. Bhattacharya, | ||
+ | A nonradioactive assay method for determination of enzymatic | ||
+ | activity of d-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). | ||
+ | Journal of Biochemical and Biophysical Methods,52(3), 179-187. | ||
+ | doi:10.1016/s0165-022x(02)00072-6</li> | ||
+ | </ol> | ||
+ | |||
+ | </div> | ||
+ | |||
</div> | </div> | ||
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Latest revision as of 03:26, 18 October 2018
Results
Hard Work Pays Off