Difference between revisions of "Part:BBa K2762013"
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We use total solution test to determine the function of CA. To view more details about the total solution test, please check the results page of 2018_NCKU_TAINAN. | We use total solution test to determine the function of CA. To view more details about the total solution test, please check the results page of 2018_NCKU_TAINAN. | ||
http://2018.igem.org/Team:NCKU_Tainan/Results | http://2018.igem.org/Team:NCKU_Tainan/Results | ||
| − | + | ===Function of CA=== | |
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 (BBa_K2762011) into pSB1C3 and cloned PRK with P<sub>lacI</sub> promoter and CA with P<sub>T7</sub> promoter(BBa_K2762013) 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 CO<sub>2</sub> concentration and thus increase the carbon flux of the bypass pathway. The efficiency of the bypass pathway is thus been increased. | 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 (BBa_K2762011) into pSB1C3 and cloned PRK with P<sub>lacI</sub> promoter and CA with P<sub>T7</sub> promoter(BBa_K2762013) 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 CO<sub>2</sub> concentration and thus increase the carbon flux of the bypass pathway. The efficiency of the bypass pathway is thus been increased. | ||
Revision as of 09:38, 17 October 2018
PlacI-B0034-prk-B0015-PT7-B0034-CcaA-B0015
Background
This composite part is composed of BBa_R0010, BBa_I719005, BBa_B0034, BBa_K2762003, BBa_K2762004, and BBa_B0015 which encodes phosphoribulokinase (PRK) and carbonic anhydrases/dehydratases (CcaA) derived from Synechococcus elongatus PCC7942 and Synechococcus elongatus PCC7002 respectively.
Characterization
Enzyme Digestion of DNA Fragement
Fig. 1 Enzyme digestion of BBa K2762013
Total Solution Test
We use total solution test to determine the function of CA. To view more details about the total solution test, please check the results page of 2018_NCKU_TAINAN. http://2018.igem.org/Team:NCKU_Tainan/Results
Function of CA
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 (BBa_K2762011) into pSB1C3 and cloned PRK with PlacI promoter and CA with PT7 promoter(BBa_K2762013) 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.
Fig. 3 Shows the growth and XUI comparison of each strain. 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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]