Oscarliu117 (Talk | contribs) |
|||
Line 66: | Line 66: | ||
<p class="pcontent"> | <p class="pcontent"> | ||
We constructed PRK fragments(<a href="http://parts.igem.org/Part:BBa_K2762007" | We constructed PRK fragments(<a href="http://parts.igem.org/Part:BBa_K2762007" | ||
− | style="color:#28ff28;">BBa_K2762007</a>) from IDT DNA synthesis. After PCR amplification, | + | style="color:#28ff28;">BBa_K2762007</a>) 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: | ||
Line 72: | Line 73: | ||
</div> | </div> | ||
− | <img class="contentimg" src=" "> | + | <img class="contentimg" src="https://static.igem.org/mediawiki/2018/8/80/T--NCKU_Tainan--Results_fig_1.jpg"> |
<div id="pt"> | <div id="pt"> | ||
Line 87: | Line 88: | ||
We initially decided to test its function by HPLC to measure the amount of RuBP | We initially decided to test its function by HPLC to measure the amount of RuBP | ||
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, determined to test its function with a toxicity test. The product of PRK-RuBP | + | 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 | ||
cannot be metabolite by wild-type <i>E. coli</i>. The accumulation of RuBP | cannot be metabolite by wild-type <i>E. coli</i>. The accumulation of RuBP | ||
depletes | depletes | ||
Line 109: | Line 111: | ||
</p> | </p> | ||
− | <img class="contentimg fig3" src="https://static.igem.org/mediawiki/2018/ | + | <img class="contentimg fig3" src="https://static.igem.org/mediawiki/2018/3/3d/T--NCKU_Tainan--Results_fig_3.png"> |
<p class="pcontent"> | <p class="pcontent"> | ||
Line 135: | Line 137: | ||
</p> | </p> | ||
− | <img class="contentimg fig4" src="https://static.igem.org/mediawiki/2018/ | + | <img class="contentimg fig4" src="https://static.igem.org/mediawiki/2018/3/3a/T--NCKU_Tainan--Results_fig_4.PNG"> |
<p class="pcontent"> | <p class="pcontent"> | ||
Line 151: | Line 153: | ||
the control group. We deduce that PRK can still function in W3110 since the | the control group. 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. The | + | expression of the protein may be lower than that of high copy number plasmid. |
+ | The | ||
pressure tolerance of W3110 strain may also lessen the toxicity influence by | pressure tolerance of W3110 strain may also lessen the toxicity influence by | ||
PRK. | PRK. | ||
</p> | </p> | ||
− | <img class="contentimg fig5" src="https://static.igem.org/mediawiki/2018/ | + | <img class="contentimg fig5" src="https://static.igem.org/mediawiki/2018/f/f6/T--NCKU_Tainan--Results_fig_4_2.PNG"> |
<p class="pcontent"> | <p class="pcontent"> | ||
Line 179: | Line 182: | ||
<p class="pcontent"> | <p class="pcontent"> | ||
We cloned the DNA fragments (<a href="http://parts.igem.org/Part:BBa_K2762008" | We cloned the DNA fragments (<a href="http://parts.igem.org/Part:BBa_K2762008" | ||
− | style="color:#28ff28;">BBa_K2762008</a>) into pSB1C3 plasmid after the gene is amplified | + | style="color:#28ff28;">BBa_K2762008</a>) 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. | ||
</p> | </p> | ||
− | <img class="contentimg" src=" "> | + | <img class="contentimg" src="https://static.igem.org/mediawiki/2018/0/04/T--NCKU_Tainan--Results_fig_5.jpg"> |
<p class="pcontent"> | <p class="pcontent"> | ||
Line 201: | Line 205: | ||
We then ran the activity test of CA. In our bypass pathway, the function of CA | We then ran the activity test of CA. In our bypass pathway, the function of CA | ||
is to | is to | ||
− | convert proton and bicarbonate into water and carbon dioxide. CA activity was determined using the Wilbur-Anderson assay. Briefly, 9 mL ice-cold Tris−HCl (20 mM, pH8.3) buffer and 0.2 mL enzyme were mixed and transferred to a 20 mL sample bottle, with further incubation at 0 °C with stirring. Then, 6 mL of ice-cold CO2-saturated solution was added immediately into the sample bottle and the time course (sec) of pH decrease from 8.3 to 6.3 was recorded. CA activity was calculated using a Wilbur–Anderson unit (WAU) per milliliter of sample. The definition for WAU is (T<sub>0</sub>-T)/(T<sub>0</sub>) in which T<sub>0</sub> and T was the time required for the pH drop from 8.3 to 6.3, with and without CA, respectively. The enzyme activity of our CA is 21.8 | + | convert proton and bicarbonate into water and carbon dioxide. CA activity was |
+ | determined using the Wilbur-Anderson assay. Briefly, 9 mL ice-cold Tris−HCl (20 | ||
+ | mM, pH8.3) buffer and 0.2 mL enzyme were mixed and transferred to a 20 mL | ||
+ | sample bottle, with further incubation at 0 °C with stirring. Then, 6 mL of | ||
+ | ice-cold CO2-saturated solution was added immediately into the sample bottle | ||
+ | and the time course (sec) of pH decrease from 8.3 to 6.3 was recorded. CA | ||
+ | activity was calculated using a Wilbur–Anderson unit (WAU) per milliliter of | ||
+ | sample. The definition for WAU is (T<sub>0</sub>-T)/(T<sub>0</sub>) in which T<sub>0</sub> | ||
+ | and T was the time required for the pH drop from 8.3 to 6.3, with and without | ||
+ | CA, respectively. The enzyme activity of our CA is 21.8 | ||
unit/liter. | unit/liter. | ||
To confirm the contribution of the CA to the whole pathway, we also ran the | To confirm the contribution of the CA to the whole pathway, we also ran the | ||
Line 228: | Line 241: | ||
</p> | </p> | ||
− | <img class="contentimg" src=" "> | + | |
+ | <img class="contentimg" src="https://static.igem.org/mediawiki/2018/c/cb/T--NCKU_Tainan--Results_fig_9.jpg"> | ||
+ | |||
+ | <p class="pcontent"> | ||
+ | Fig. 8 Confirmation of rbcX and rbcS digestion | ||
+ | |||
+ | Fig. 9 Confirmation of RbcX and RbcS expression in BL21(DE3) The expected protein | ||
+ | size is 15.3 kDA and 13.8kDA respectively. | ||
+ | </p> | ||
+ | |||
+ | <img class="contentimg" src="https://static.igem.org/mediawiki/2018/0/0d/T--NCKU_Tainan--Results_fig_7.jpg"> | ||
<p class="pcontent"> | <p class="pcontent"> | ||
− | Fig. | + | Fig. 10 Confirmation of rbcL digestion.</br> |
− | Fig. | + | Fig. 11 Confirmation of rbcL expression in DH5˚α. The expected protein size is |
52.37kDa.</br> | 52.37kDa.</br> | ||
</p> | </p> | ||
Line 286: | Line 309: | ||
impractical for us since we have too much test samples. A new method to measure | impractical for us since we have too much test samples. A new method to measure | ||
multiple samples in the short period of time is developed by our team. We are | multiple samples in the short period of time is developed by our team. We are | ||
− | able to evaluate the fixation efficiency of each sample with the optical density | + | able to evaluate the fixation efficiency of each sample with the optical |
+ | 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 enzyme of our construction is necessary for carbon fixation. | that the enzyme of our construction is necessary for carbon fixation. | ||
Line 317: | Line 341: | ||
</ol> | </ol> | ||
− | <img class="contentimg fig10" src="https://static.igem.org/mediawiki/2018/ | + | <img class="contentimg fig10" src="https://static.igem.org/mediawiki/2018/6/64/T--NCKU_Tainan--Results_fig_10.png"> |
<p class="pcontent"> | <p class="pcontent"> | ||
− | Fig. | + | Fig. 12 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. | ||
Line 347: | Line 371: | ||
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 | ||
− | producing the same amount of carbon biomass, it requires less xylose. We can thus | + | producing the same amount of carbon biomass, it requires less xylose. We can |
+ | thus | ||
compare the XUI of each strain to determine the possible strain that fix | compare the XUI of each strain to determine the possible strain that fix | ||
carbon. | carbon. | ||
Line 368: | Line 393: | ||
<p class="pcontent"> | <p class="pcontent"> | ||
− | Fig. | + | Fig. 13 Shows the calibration line of DNS assay kit. |
</p></br> | </p></br> | ||
Line 386: | Line 411: | ||
<p class="pcontent"> | <p class="pcontent"> | ||
− | Fig. | + | Fig. 14 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 show growth after 24hours. | strains show growth after 24hours. | ||
Line 415: | Line 440: | ||
</p> | </p> | ||
− | <img class="contentimg col-6" src="https://static.igem.org/mediawiki/2018/ | + | <img class="contentimg col-6" src="https://static.igem.org/mediawiki/2018/3/35/T--NCKU_Tainan--Results_fig_13.png"> |
− | <img class="contentimg col-6" src="https://static.igem.org/mediawiki/2018/ | + | <img class="contentimg col-6" src="https://static.igem.org/mediawiki/2018/0/0e/T--NCKU_Tainan--Results_fig_14.png"> |
<p class="pcontent"> | <p class="pcontent"> | ||
− | Fig. | + | Fig. 15 Shows the growth and XUI measured in 5% CO<sub>2</sub> incubation of 12 |
hours | 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 | ||
Line 447: | Line 472: | ||
</p> | </p> | ||
− | <img class="contentimg col-6" src="https://static.igem.org/mediawiki/2018/ | + | <img class="contentimg col-6" src="https://static.igem.org/mediawiki/2018/9/99/T--NCKU_Tainan--Results_fig_15.png"> |
− | <img class="contentimg col-6 fig14_2" src="https://static.igem.org/mediawiki/2018/ | + | <img class="contentimg col-6 fig14_2" src="https://static.igem.org/mediawiki/2018/5/54/T--NCKU_Tainan--Results_fig_16.png"> |
<p class="pcontent"> | <p class="pcontent"> | ||
− | Fig. | + | Fig. 16 Shows the growth and XUI comparison of each strain. All the tested |
strains are incubated in 5% CO<sub>2</sub> incubator for 12 hr. 0.1mM of IPTG | strains are incubated in 5% CO<sub>2</sub> incubator for 12 hr. 0.1mM of IPTG | ||
was added to | was added to | ||
Line 492: | Line 517: | ||
</ol> | </ol> | ||
− | <img class="contentimg col-6" src="https://static.igem.org/mediawiki/2018/ | + | <img class="contentimg col-6" src="https://static.igem.org/mediawiki/2018/0/0c/T--NCKU_Tainan--Results_fig_17.png"> |
− | <img class="contentimg col-6 fig14_2" src="https://static.igem.org/mediawiki/2018/ | + | <img class="contentimg col-6 fig14_2" src="https://static.igem.org/mediawiki/2018/5/52/T--NCKU_Tainan--Results_fig_18.png"> |
<p class="pcontent"> | <p class="pcontent"> | ||
− | Fig. | + | Fig. 17 Shows the growth and the XUI of BL21(DE3) and W3110 strains. |
</p></br> | </p></br> | ||
Line 523: | Line 548: | ||
<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="https://static.igem.org/mediawiki/2018/6/67/T--NCKU_Tainan--Results_Fig_15.PNG"> | ||
− | <img class="contentimg col-6 fig16_2" src="https://static.igem.org/mediawiki/2018/ | + | <img class="contentimg col-6 fig16_2" src="https://static.igem.org/mediawiki/2018/7/71/T--NCKU_Tainan--Results_fig_20.png"> |
<p class="pcontent"> | <p class="pcontent"> | ||
− | Fig. | + | 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 was grown in | strain was grown in | ||
Line 692: | Line 717: | ||
<div id="pt"> | <div id="pt"> | ||
<p class="pcontent"> | <p class="pcontent"> | ||
− | P<sub>asr</sub> is reported to be induce in acidic condition. We think that in can be used | + | P<sub>asr</sub> 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 | 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 | fluorescent intensity in a short period of time. We first incubated the | ||
Line 704: | Line 730: | ||
</p> | </p> | ||
− | <img class="contentimg" src=""> | + | <img class="contentimg" src="https://static.igem.org/mediawiki/2018/4/44/T--NCKU_Tainan--Results_fig_21.png"> |
<p class="pcontent"> | <p class="pcontent"> | ||
Line 733: | Line 759: | ||
</p> | </p> | ||
− | <img class="contentimg" src=""> | + | <img class="contentimg" src="https://static.igem.org/mediawiki/2018/a/ac/T--NCKU_Tainan--Results_fig_22.png"> |
<p class="pcontent"> | <p class="pcontent"> | ||
Line 745: | Line 771: | ||
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>. For more information, please check | + | riboJ sequence at the downstream of P<sub>gadA</sub>. For more information, |
+ | please check | ||
the Improvement page. | the Improvement page. | ||
</p> | </p> | ||
− | </br><h3>References</h3> | + | </br> |
− | + | <h3>References</h3> | |
+ | |||
<ol> | <ol> | ||
<li class="smallp">Gong, F., Liu, G., Zhai, X., Zhou, J., Cai, Z., & Li, Y. | <li class="smallp">Gong, F., Liu, G., Zhai, X., Zhou, J., Cai, Z., & Li, Y. |
Revision as of 03:01, 16 October 2018
Results