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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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<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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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 | ||
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$${=\ 0.575\ {mg\over L ∙hr}}$$ | $${=\ 0.575\ {mg\over L ∙hr}}$$ | ||
− | 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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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. | ||
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<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. | ||
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style="color:#28ff28;">BBa_K1962013</a>) was previously reported to be induced under neutral and mild | 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<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"> |
Latest revision as of 03:26, 18 October 2018
Results
Hard Work Pays Off