Oscarliu117 (Talk | contribs) |
Oscarliu117 (Talk | contribs) |
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measurement such as excessive noise signal in our sample.We therefore | measurement such as excessive noise signal in our sample.We therefore | ||
determined to test its function with a toxicity test. The product of PRK-RuBP | determined to test its function with a toxicity test. The product of PRK-RuBP | ||
− | cannot be metabolite by wild type <i>E. coli</i>. The accumulation of RuBP depletes | + | cannot be metabolite 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 | ||
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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> strains</li> | + | <li>Compare the carbon fixation rate of W3110 and BL21(DE3) <i>E. coli</i> |
+ | strains</li> | ||
<li>Compare different CO<sub>2</sub> incubation environment</li> | <li>Compare different CO<sub>2</sub> incubation environment</li> | ||
Line 342: | Line 344: | ||
consumption over O.D. 600. O.D. 600 measurement can be viewed as the weight of | 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 | 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 | + | 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> | provided in our medium) as its carbon source. Although some native <i>E. coli</i> | ||
pathway | pathway | ||
− | may utilize CO<sub>2</sub> (such as lipid synthesis), the amount is too small to consider. | + | may utilize CO<sub>2</sub> (such as lipid synthesis), the amount is too small |
+ | to consider. | ||
As | As | ||
for engineered strain, carbon dioxide can be utilized as it’s carbon source. By | for engineered strain, carbon dioxide can be utilized as it’s carbon source. By | ||
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strain and discovered that control strain without IPTG induction produce less | strain and discovered that control strain without IPTG induction produce less | ||
rubisco enzyme than the experiment. Without rubisco, the bypass pathway is not | rubisco enzyme than the experiment. Without rubisco, the bypass pathway is not | ||
− | capable of using CO<sub>2</sub>. We found out that the strain without Rubisco has higher | + | capable of using CO<sub>2</sub>. We found out that the strain without Rubisco |
+ | has higher | ||
XUI, symbolizing that rubisco is essential in carbon fixation pathway. | XUI, symbolizing that rubisco is essential in carbon fixation pathway. | ||
</p> | </p> | ||
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<p class="pcontent"> | <p class="pcontent"> | ||
− | Fig. 13 Shows the growth and XUI measured in 5% CO<sub>2</sub> incubation of 12 hours | + | Fig. 13 Shows the growth and XUI measured in 5% CO<sub>2</sub> incubation of 12 |
+ | hours | ||
respectively. Lower growth of the strain that contains. The XUI of the strain | respectively. Lower growth of the strain that contains. 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 | ||
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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 CO<sub>2</sub> concentration and thus increase | + | 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. | ||
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<p class="pcontent"> | <p class="pcontent"> | ||
Fig. 14 Shows the growth and XUI comparison of each strains. All the tested | Fig. 14 Shows the growth and XUI comparison of each strains. All the tested | ||
− | strains are incubated in 5% CO<sub>2</sub> incubator for 12 hr. 0.1mM of IPTG was added to | + | strains are incubated in 5% CO<sub>2</sub> incubator for 12 hr. 0.1mM of IPTG |
+ | was added to | ||
induce the protein expression. We can observe that growth speed of the | induce the protein expression. We can observe that growth speed of the | ||
construction has been increased with the CA, and the XUI of the strain that | construction has been increased with the CA, and the XUI of the strain that | ||
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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 <i>E. coli</i> strain. We found out that both strain | + | the difference of XUI between two <i>E. coli</i> strain. We found out that both |
+ | 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. HoweverW3110 has a higher XUI compared with BL21(DE3) in | constructed protein. HoweverW3110 has a higher XUI compared with BL21(DE3) in | ||
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<p class="pcontent"> | <p class="pcontent"> | ||
− | Finally, we compare the XUI under different CO<sub>2</sub> concentration. We incubated the | + | Finally, we compare the XUI under different CO<sub>2</sub> concentration. We |
− | bacteria in normal incubator without CO<sub>2</sub> input and the cell culture incubator | + | incubated the |
− | that maintains 5% CO<sub>2</sub> concentration. We observed that the strain in 5% CO<sub>2</sub> | + | bacteria in normal incubator without CO<sub>2</sub> input and the cell culture |
− | incubator has lower the XUI. The supply of sufficient CO<sub>2</sub> can increase the | + | incubator |
+ | that maintains 5% CO<sub>2</sub> concentration. We observed that the strain in | ||
+ | 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 concluded that | efficiency of the bypass pathway and enhance the growth. We can concluded that | ||
our constructed pathway can be utilize carbon dioxide as one of its carbon | our constructed pathway can be utilize carbon dioxide as one of its carbon | ||
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<p class="pcontent"> | <p class="pcontent"> | ||
Fig. 16 The comparison of the growth and the XUI of the BL21(DE3) that contains | Fig. 16 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 strain grown in | + | all three enzymes in normal incubator and 5% CO<sub>2</sub> incubator. The |
− | CO<sub>2</sub> incubator shows better growth and lower XUI, which indicates that our | + | strain grown in |
− | strain can use CO<sub>2</sub> as a carbon source in the presence of high CO<sub>2</sub> level. | + | CO<sub>2</sub> incubator shows 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> | ||
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<div id="pt"> | <div id="pt"> | ||
<p class="pcontent"> | <p class="pcontent"> | ||
− | To find out how much and how efficient genetically engineered <i>E. coli</i> can fix | + | 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 | 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 | + | heterotrophic CO<sub>2</sub> fixation process. Consider a system composed of a |
+ | single | ||
component, the general material balance can be written as: | component, the general material balance can be written as: | ||
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=\ \{C_{biomass}\}...(1)}$$ | =\ \{C_{biomass}\}...(1)}$$ | ||
− | Considering the difficulties in measuring carbon in <i>E. coli</i> metabolic waste and | + | 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 | that C<sub>waste</sub> would be positive, the equation reduces to | ||
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<p class="pcontent"> | <p class="pcontent"> | ||
C<sub>biomass</sub> can be calculate by multiplying O.D. 600 to DCW and mass | 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 | + | 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 | 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 | Li et al. reported that dry cell weight (DCW) of <i>E. coli</i> is | ||
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$${0.35g\over L ∙ 𝑂.𝐷. 600}$$ | $${0.35g\over L ∙ 𝑂.𝐷. 600}$$ | ||
− | , determined by experiment. <i>E. coli</i> biomass contains 48% of carbon by mass. | + | , determined by experiment. <i>E. coli</i> biomass contains 48% of carbon by |
+ | mass. | ||
$${C_{biomass}\ =\ 0.45\ ×\ 0.35\ ×\ 48\%}$$ | $${C_{biomass}\ =\ 0.45\ ×\ 0.35\ ×\ 48\%}$$ | ||
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$${=\ 0.0066704\ g/L}$$ | $${=\ 0.0066704\ g/L}$$ | ||
− | Since the <i>E. coli</i> has been cultured for 12 hours, we can calculate the rate of | + | Since the <i>E. coli</i> has been cultured for 12 hours, we can calculate the |
+ | rate of | ||
carbon fixation by | carbon fixation by | ||
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$${=\ 0.5558\ {mg\over L ∙hr}}$$ | $${=\ 0.5558\ {mg\over L ∙hr}}$$ | ||
− | To find out how much carbon in biomass comes from the carbon in CO<sub>2</sub> captured by | + | To find out how much carbon in biomass comes from the carbon in CO<sub>2</sub> |
+ | captured by | ||
the heterotrophic microbes, divide the net amount of carbon fixed by the mass | the heterotrophic microbes, divide the net amount of carbon fixed by the mass | ||
percent of carbon in biomass. | percent of carbon in biomass. | ||
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$${=\ 8.82\%}$$ | $${=\ 8.82\%}$$ | ||
</p> | </p> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | |||
+ | </div> | ||
+ | |||
+ | |||
+ | <div id="Carbon_fixation"> | ||
+ | </br></br></br></br> | ||
+ | <h3>pH sensing system</h3> | ||
+ | |||
+ | <h8>Achievements:</h8> | ||
+ | |||
+ | <ol> | ||
+ | |||
+ | <li>Construct the pH sensing system</li> | ||
+ | |||
+ | <li>Measure the short-term fluorescent intensity of P<sub>asr</sub></li> | ||
+ | |||
+ | <li>Measure the long-term fluorescent intensity of P<sub>gadA</sub></li> | ||
+ | |||
+ | </ol></br> | ||
+ | |||
+ | <h8>Construction of the pH sensing system</h8> | ||
+ | |||
+ | <div id="pt"> | ||
+ | <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 rbc 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> | ||
+ | |||
+ | <h8>Fluorescent intensity measurement of P<sub>asr</sub></h8> | ||
+ | |||
+ | <div id="pt"> | ||
+ | <p class="pcontent"> | ||
+ | Pasr is reported to be induce in acidic condition. We think that in can be used | ||
+ | to report the abnormal acidity of the medium. We thus determine to measure the | ||
+ | fluorescent 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 fluorescent intensity for every 3 minutes. We found out that the | ||
+ | promoter Pasr will be induced at the pH value below four within 30 minutes. The | ||
+ | different fluorescent intensity can be observed within 30 minutes. The | ||
+ | fluorescent had the peak at pH value of 4.25. | ||
+ | </p> | ||
+ | |||
+ | <img class="contentimg" src=""> | ||
+ | |||
+ | <p class="pcontent"> | ||
+ | Fig. 17 The data shows the fluorescent intensity (absorbance: 485 nm, | ||
+ | excitation: | ||
+ | 535 nm) expressed by Pasr in different pH. | ||
+ | </p></br> | ||
+ | |||
+ | <p class="pcontent"> | ||
+ | Based on the data has shown above, we could conclude that Pasr is an acidic | ||
+ | promoter as it has a high expression of fluorescent at pH 4.25 and pH 5. The | ||
+ | results show that Pasr constructed pH sensing system can be used as an alert. | ||
+ | When the medium turns acidic, fluorescent can be easily observed. We believe | ||
+ | that this system can also be applied to various bio-detection system. | ||
+ | </p> | ||
+ | |||
+ | <h8>Fluorescent intensity measurement of P<sub>gadA</sub></h8> | ||
+ | |||
+ | <p class="pcontent"> | ||
+ | P<sub>gadA</sub> was previously reported to be induced under neutral and mild | ||
+ | acidic | ||
+ | environment. We measure the fluorescent 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 | ||
+ | mild expression. | ||
+ | </p> | ||
+ | |||
+ | <img class="contentimg" src=""> | ||
+ | |||
+ | <p class="pcontent"> | ||
+ | Fig. 18 The data shows the fluorescent intensity (absorbance: 485 nm, | ||
+ | excitation: 535 nm) expressed by PGadA in different pH. | ||
+ | </p> | ||
+ | |||
+ | <p class="pcontent"> | ||
+ | We found out that the fluorescent 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 PGadA. For more information, please check | ||
+ | the Improvement page. | ||
+ | </p> | ||
+ | |||
</div> | </div> | ||
Revision as of 15:49, 15 October 2018
Results