Oscarliu117 (Talk | contribs) |
Oscarliu117 (Talk | contribs) |
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incubator for 24 hours. The growth of W3110(L5T7) is not obvious while other | incubator for 24 hours. The growth of W3110(L5T7) is not obvious while other | ||
strains shows growth after 24hours. | strains shows growth after 24hours. | ||
− | </p> | + | </p></br> |
− | <h8>Total solution check: Function of Rubisco</h8> | + | <h8>Total solution check: Function of Rubisco</h8></br> |
<p class="pcontent"> | <p class="pcontent"> | ||
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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> | + | 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 | |
− | + | plasmids were then co-transformed into BL21(DE3). We | |
− | + | measure the XUI of the strain and compare to the control that IPTG was not | |
− | + | 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 | |
− | + | 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 | rubisco. The control strain without IPTG induction produce less rubisco enzyme | ||
− | than | + | than the experiment and had less pressure. We then compare the XUI of each |
− | + | strain and discovered that control strain without IPTG induction produce less | |
− | and | + | rubisco enzyme than the experiment. Without rubisco, the bypass pathway is not |
− | + | capable of using CO2. We found out that the strain without Rubisco has higher | |
− | enzyme | + | XUI, symbolizing that rubisco is essential in carbon fixation pathway. |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
</p> | </p> | ||
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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. | compare to strain without both enzymes. | ||
+ | </p> | ||
+ | |||
+ | <h8>Total solution check: Function of CA</h8> | ||
+ | |||
+ | <p class="pcontent"> | ||
+ | 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 | ||
+ | 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 | ||
+ | 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>) | ||
+ | into pSB3K3. Two plasmids are then co-transformed into BL21(DE3). We measured | ||
+ | the | ||
+ | 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 | ||
+ | infer that CA can enhance the intracellular CO2 concentration and thus increase | ||
+ | the carbon flux of the bypass pathway. The efficiency of the bypass pathway is | ||
+ | thus been increased. | ||
+ | </p> | ||
+ | |||
+ | <img class="contentimg col-6" src=" "> | ||
+ | |||
+ | <img class="contentimg col-6" src=" "> | ||
+ | |||
+ | <p class="pcontent"> | ||
+ | Fig. 13 Shows the growth and XUI comparison of each strains. All the tested | ||
+ | strains are incubated in 5% CO2 incubator for 12 hr. 0.1mM of IPTG was added to | ||
+ | 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 | ||
+ | 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 | ||
+ | enzymes are required to optimized the carbon fixing bypass pathway. | ||
</p> | </p> | ||
Revision as of 14:39, 12 October 2018
Results