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<p class="pcontent"> | <p class="pcontent"> | ||
− | We constructed PRK fragments | + | We constructed PRK fragments from IDT DNA synthesis. After PCR amplification, |
− | + | ||
PRK is then cloned into pSB1C3 and transformed into DH5α. SDS-PAGE ensured that | PRK is then cloned into pSB1C3 and transformed into DH5α. 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: | ||
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</p> | </p> | ||
− | <img class="contentimg" src=" "> | + | <img class="contentimg" src="https://static.igem.org/mediawiki/2018/9/9f/T--NCKU_Tainan--Results_Fig_3.PNG"> |
<p class="pcontent"> | <p class="pcontent"> | ||
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</p> | </p> | ||
− | <img class="contentimg" src=" "> | + | <img class="contentimg" src="https://static.igem.org/mediawiki/2018/5/5e/T--NCKU_Tainan--Results_Fig_4.PNG"> |
<p class="pcontent"> | <p class="pcontent"> | ||
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</p> | </p> | ||
− | <img class="contentimg" src=" "> | + | <img class="contentimg" src="https://static.igem.org/mediawiki/2018/8/83/T--NCKU_Tainan--Results_Fig_4_W3110.PNG"> |
+ | <p class="pcontent"> | ||
+ | Fig. 5 The result of PRK test in W3110 | ||
+ | </p></br> | ||
</div> | </div> | ||
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<p class="pcontent"> | <p class="pcontent"> | ||
− | We cloned the DNA fragments | + | We cloned the DNA fragments into pSB1C3 plasmid after the gene is amplified |
− | + | ||
with PCR. We transform the plasmid into DH5-alpha and BL21(DE3). Next, we | 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. | ||
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<p class="pcontent"> | <p class="pcontent"> | ||
− | Fig. | + | Fig. 6 Confirmation of ca 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> | ||
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<div id="pt"> | <div id="pt"> | ||
<p class="pcontent"> | <p class="pcontent"> | ||
− | + | We then ran the activity test of CA. In our bypass pathway, the function of CA | |
+ | is to | ||
+ | convert proton and bicarbonate into water and carbon dioxide. During the | ||
+ | reaction, the | ||
+ | concentration of proton can be easily observed by the pH meter. By measuring | ||
+ | the time | ||
+ | interval between the pH shift and compare with the sample without CA, we can | ||
+ | determine | ||
+ | the activity of that specific enzyme. The enzyme activity of our CA is 21.8 | ||
+ | unit/liter. | ||
+ | To confirm the contribution of the CA to the whole pathway, we also ran the | ||
+ | total | ||
+ | solution which will be described in the following content. | ||
</p> | </p> | ||
</div></br> | </div></br> | ||
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<p class="pcontent"> | <p class="pcontent"> | ||
− | Fig. | + | Fig. 8 Confirmation of rbcL digestion.</br> |
− | Fig. | + | Fig. 9 Confirmation of rbcL expression in DH5˚α. The expected protein size is |
52.37kDa.</br> | 52.37kDa.</br> | ||
</p> | </p> | ||
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</ol> | </ol> | ||
− | <img class="contentimg" src=" "> | + | <img class="contentimg" src="https://static.igem.org/mediawiki/2018/f/f2/T--NCKU_Tainan--Results_Fig_9.PNG"> |
<p class="pcontent"> | <p class="pcontent"> | ||
− | Fig. | + | Fig. 10 shows the dry cell weight of BL21(DE3) incubated in altered M9 xylose |
medium. | medium. | ||
A linear relationship between O.D. and dry cell weight is observed. | A linear relationship between O.D. and dry cell weight is observed. | ||
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</p> | </p> | ||
− | <img class="contentimg" src=" "> | + | <img class="contentimg" src="https://static.igem.org/mediawiki/2018/3/3f/T--NCKU_Tainan--Results_Fig_10.PNG"> |
<p class="pcontent"> | <p class="pcontent"> | ||
− | Fig. | + | Fig. 11 Shows the calibration line of DNS assay kit. |
</p></br> | </p></br> | ||
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− | <img class="contentimg" src=" "> | + | <img class="contentimg" src="https://static.igem.org/mediawiki/2018/5/51/T--NCKU_Tainan--Results_Fig_11.PNG"> |
<p class="pcontent"> | <p class="pcontent"> | ||
− | Fig. | + | Fig. 12 shows the growth of W3110(L5T7), BL21(DE3), W3110 incubated in normal |
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. | ||
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</p> | </p> | ||
− | <img class="contentimg col-6" src=" "> | + | <img class="contentimg col-6" src="https://static.igem.org/mediawiki/2018/9/9d/T--NCKU_Tainan--Results_Fig_12.PNG"> |
− | <img class="contentimg col-6" src=" "> | + | <img class="contentimg col-6" src="https://static.igem.org/mediawiki/2018/d/dd/T--NCKU_Tainan--Results_Fig_12_2.PNG"> |
<p class="pcontent"> | <p class="pcontent"> | ||
− | Fig. | + | Fig. 13 Shows the growth and XUI measured in 5% CO2 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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</p> | </p> | ||
− | <img class="contentimg col-6" src=" "> | + | <img class="contentimg col-6" src="https://static.igem.org/mediawiki/2018/4/47/T--NCKU_Tainan--Results_Fig_14.PNG"> |
− | <img class="contentimg col-6" src=" "> | + | <img class="contentimg col-6" src="https://static.igem.org/mediawiki/2018/0/0a/T--NCKU_Tainan--Results_Fig_14_2.PNG"> |
<p class="pcontent"> | <p class="pcontent"> | ||
− | Fig. | + | Fig. 14 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 | 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 | induce the protein expression. We can observe that growth speed of the | ||
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<p class="pcontent"> | <p class="pcontent"> | ||
− | Fig. | + | Fig. 15 Shows the growth and the XUI of BL21(DE3) and W3110 strains. |
</p></br> | </p></br> | ||
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</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" src="https://static.igem.org/mediawiki/2018/4/42/T--NCKU_Tainan--Results_Fig_15_2.PNG"> |
<p class="pcontent"> | <p class="pcontent"> | ||
− | Fig. | + | Fig. 16 The comparison of the growth and the XUI of the BL21(DE3) that contains |
all three enzymes in normal incubator and 5% CO2 incubator. The strain grown in | all three enzymes in normal incubator and 5% CO2 incubator. The strain grown in | ||
CO2 incubator shows better growth and lower XUI, which indicates that our | CO2 incubator shows better growth and lower XUI, which indicates that our |
Revision as of 14:55, 13 October 2018
Results