Part:BBa_K2762011

PT7-B0034-rbcL-B0015-PT7-B0034-rbcX-B0034-rbcS-B0015

Background

Ribulose-1,5-biphosphate carboxylase/oxygenase catalyzes the first reaction of the Calvin cycle, converting the combination of Ribulose-1,5-biphosphate(RuBP) and carbon dioxide and then form two 3-phosphoglycerate molecular. 3-phophoglycerate will then convert to pyruvate by the native metabolic system of E coli. Previous studies have utilized E. coli as a host of studying point mutation and random mutation of RubisCO in order to enhance its enzyme activity. This study proved that installing rubisco gene into E. coli system is actually feasibly. After mining information from journal, we selected rubisco gene form Synechococcus elongtus PCC7002, which is a well-studied cyanobacteria.

Structure and the Function of the protein

The structure of RubisCO involves two polypeptide subunit: RbcL and RbcS. Each RubisCO is consist of eight RbcL and RbcS. RbcL, the large chain of RubisCO, contains the main active site. The active site requires Mg₂₊ ion. The ion binds to the certain amino acid of the central of RbcL, activate the RubisCo. Since the M9 medium contains Mg2+ ion, we only need to add little amount of additional ion, adjusting the concentration to 20mM.

It has reported that RbcL can be functional solely. However, researches have revealed that RbcS contribute to the activity and the CO₂/O₂ specification through the mutagenesis and hybridization of cyanobacteria RubisCO, and indicated the RbcS should take into consideration while constructing the Calvin cycle. For this reason, we decide to add RbcS gene to our construction.

RbcX, the subunit which doesn’t involve in the structure of RubisCO, however, plays an important role in the structure folding. Researches have revealed that without the presence of the RbcX chaperon, RubisCO cannot fold correctly. According to these researches, we take the RbcX into our consideration and add the gene into our construction.

Characterization

We did three experiments to confirm the expression and function of this part. First, we inserted the part on pSB1C3 and cloned the plasmid into DH5 alpha. We extracted the plasmid after the colony formed and did the enzyme digestion to confirm the insertion was successful. Second, we did the SDS-PAGE to confirm the presence of each polypeptide. Third, we cultured the BL21(DE3) with the T7-RubisCO part and PRK part in the M9-xylose medium in 5% CO₂ incubator and normal incubator after inducing the RubisCo gene with IPTG. We also did the non-induced control groups. The result showed that the with the presence of these two functional enzymes. The E. coli grew faster and consumed more xylose in the CO₂ rich environment.

Colony PCR of the construct

After finishing the rbc biobrick construction, colony PCR is run to check the success of ligation. The length of the DNA is verified with agarose gel electrophoresis.

Fig.1 shows the colony PCR of BBa K2762011.

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 Rubisco

We then utilized XUI to evaluate the function of each enzyme in the pathway. We first check the function of Rubisco in BL21(DE3) strain. Rubisco enzyme with promoter PT7 (BBa_K2762011) was cloned into pSB1C3 and PRK with promoter P_lacI (BBa_K2762007)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 PT7 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 RubisCO. The control strain without IPTG induction produce less rubisco enzyme 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 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 XUI, symbolizing that rubisco is essential in carbon fixation pathway.

Fig. 2 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 that contains both Rubisco and PRK shows statistically significant decrease compare to strain without both enzymes.

Comparison between BL21(DE3) and W3110

We then compare the XUI value between BL21(DE3) and W3110 constructed strain. When we design our IDT sequence, we link the CA directly to the promoter P_lacI, so we could not transform CA construct into W3110 strain. We thus compare the XUI of strains that only contains Rubisco and PRK (BBa_K2762011) , (BBa_K2762007) . We found out that both strains shows a similar trend: the XUI will be lower with the expression of the constructed protein. The growth condition of both constructed strains is similar for the first 12 hours. We then compare the difference of XUI between two E. coli strain. We found out that both strains shows a similar trend: the XUI will be lower with the expression of the constructed protein. HoweverW3110 has a higher XUI compared with BL21(DE3) in constructed strain as well as the strain without plasmid. We infer two reasons that cause the difference of XUI: 1. W3110 “wildtype” strain has a more flexible metabolic network but consumes more xylose compare to lab strains such as BL21(DE3). 2. The constructed protein expression in W3110 may be less than BL21(DE3) lab strain. BL21(DE3) commonly used to express the protein. We inferred that with more protein been expressed, the bypass pathway in BL21(DE3) will be more favored than the W3110 strain.

Fig. 3 Shows the growth and the XUI of BL21(DE3) and W3110 strains.

We finally concluded that the efficiency of the bypass pathway in BL21(DE3) is better than that in the W3110 strain.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1198
  Illegal AgeI site found at 301
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

[1] Janet Newman 1t and Steven Gutteridge2*. (1994,JUNE.15). Structure of an effector-induced inactivated state of ribulose 1,5-bisphosphate carboxylase/oxygenase: the binary complex between enzyme and xylulose 1,5-bisphosphate. cell. Doi:https://doi.org/10.1016/S0969-2126(00)00050-2

[2] Inger Andersson ^a,*, Anders Backlund ^b. (2007, DEC.20). Structure and function of Rubisco. Plant Physiology and Biochemistry. Doi:10.1016/j.plaphy.2008.01.001

[3] Fuyu Gong, Guoxia Liu, Xiaoyun Zhai,Jie Zhou, Zhen Cai and Yin Li1 .(2015,Jun 18). Quantitative analysis of an engineered CO₂-fixing Escherichia coli reveals great potential of heterotrophic CO₂ fixation. Biotechnology for Biofuels.