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<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> | ||
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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 | ||
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<p class="pcontent"> | <p class="pcontent"> | ||
− | Fig 4. Compares the growth in M9 xylose medium of PRK expressing strain | + | 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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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> | ||
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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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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. | ||
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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> | ||
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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. | + | 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: | ||
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<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> | ||
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<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 | ||
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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 | ||
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<div id="pt"> | <div id="pt"> | ||
<p class="pcontent"> | <p class="pcontent"> | ||
− | The engineered <i>E. coli</i> BL21 (DE3) | + | 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 | ||
Line 677: | Line 676: | ||
$${=\ 0.575\ {mg\over L ∙hr}}$$ | $${=\ 0.575\ {mg\over L ∙hr}}$$ | ||
− | To find out how much carbon in biomass comes from the carbon in | + | 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 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> | ||
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<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> | ||
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<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 737: | 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 751: | 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 762: | 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> | ||
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</p> | </p> | ||
<br> | <br> | ||
+ | <h8>Improvement of P<sub>gadA</sub></h8></br></br> | ||
+ | |||
<p class="pcontent"> | <p class="pcontent"> | ||
We found out that the fluorescence intensity of P<sub>gadA</sub> is much lower | We found out that the fluorescence intensity of P<sub>gadA</sub> is much lower | ||
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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 | + | 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"> | <img class="contentimg fig3" src="https://static.igem.org/mediawiki/2018/f/fd/T--NCKU_Tainan--PGADA_OMPARISON.png"> | ||
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Fig 21. The data compares the fluorescence of P<sub>gadA</sub> and P<sub>gadA</sub> with RiboJ sequence. | Fig 21. The data compares the fluorescence of P<sub>gadA</sub> and P<sub>gadA</sub> with RiboJ sequence. | ||
</p> | </p> | ||
− | + | <br> | |
<p class="pcontent">For more information, | <p class="pcontent">For more information, | ||
please check | please check |
Latest revision as of 03:26, 18 October 2018
Results
Hard Work Pays Off