Part:BBa_K2762013

P_lacI-B0034-prk-B0015-P_T7-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 P_lacI promoter and CA with P_T7 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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Reference

[1]M. Wilbur, N.G. Anderson.(1948, Oct.1) Electrometric and colorimetric determination of carbonic anhydrase, J. Biol. Chem. 147–154.

[2] Lindskog S. (1997) .Structure and mechanism of carbonic anhydrase. Pharmacol Ther.

[3] Rowlett RS. (2010,Feb). Structure and catalytic mechanism of the β-carbonic anhydrases.Biochimica et Biophysica Acta (BBA)

[4] Fuyu Gong1,2, Guoxia Liu1, Xiaoyun Zhai1,2, Jie Zhou1, Zhen Cai1* and Yin Li1 .(2015,Jun 18). Biotechnology for BiofuelsQuantitative analysis of an engineered CO₂-fixing Escherichia coli reveals great potential of heterotrophic CO₂ fixation