Overview of Designed pathway

Calvin-Benson cycle is one of the most important pathways for inorganic carbon to transfer to organic carbon in carbon cycle. Plant, algae, and cyanobacteria utilize light as energy source for Calvin-Benson cycle. Taking the advantage of the pentose phosphate pathway, native metabolic pathway E. coli, it only requires two enzymes to complete the pathway-- prk and rubisco, which we will give some more detailed introduction below. The primary product of the pathway is pyruvate, which can be utilized to produce various valuable products.

Chassis Organism

We would like to test our pathway in various E. coli strains to see the functionality of our construction. We select three different strains: BL21 (DE3), W3110, W3110 (L5T7). BL21 (DE3) is a common expression B strain that is widely used to express the recombinant protein with T7 polymerase. We expected that the high production of protein may change the entire native metabolic pathway of it. W3110 (K-12 sild type strain) is reported to have good growth in stressed environment. We expected that W3110 will grow well even the sugar source is xylose. W3110(L5T7) is a constructed lab strain based on W3110. T7 polymerase was inserted to.

Prk

What is its function?

We first introduced phosphoribulokinase (PRK), an enzyme from cyanobacterial Calvin cycle, into the central carbon metabolic pathway of E. coli. PRK catalyzes the conversion of ribulose-5-phosphate (Ru5P) from the pentose phosphate pathway of the central carbon metabolism to ribulose-1,5-biphosphate (RuBP). The accumulation of RuBP in E. coli would cause cell growth arrest because E. coli could not metabolize RuBP. Another enzyme from the cyanobacterial Calvin cycle, Rubisco, is therefore needed to convert RuBP into 3-phosphogycerate (3PGA), a substance involved in glycolysis of the central metabolism pathway.

How we constructed the design?

We initially confirm the gene sequence of Synechococcus elongtus prk from NCBI gene database. We then codon optimized the sequence so E. coli can express the protein properly. The optimized sequence was sent to IDT for gene synthesis. We PCR amplified the gene fragments and digest it with HindIII and SpeI. After digestion, we ligate the fragments into Psb3k3 plasmid with P_Lac-rbs(B0034) in the upstream of fragment. The basic parts is then transformed into DH5 alpha to conserve the plasmid for the following construction.

How we test its function?

We initially decided to measure the concentration of RuBP by HPLC. Our instructors pointed out some difficulties of HPLC measurement such as too much noise signal in our sample. We then design the experiment to prove the function of prk in an indirect manner: measuring its growth rate. RuBP, the product of prk is toxic to E. coli. We express this protein independently in xylose M9 to check the cell growth. If the cell growth is arrested, we can indirectly conclude the function of prk.

Rubisco

What is its function?

Ribulose-1,5-biphosphate carboxylase/oxygenase is one of the world’s most abundant enzyme. It catalyzes the conversion of inorganic carbon into organic carbon. In our designed pathway, the function of the rubisco is to convert Ribulose-1,5-biphosphate (RuBP) from the upper pathway and carbon dioxide into 3-phosphoglycerate (3PGA). 3PGA will then convert to pyruvate by the native metabolic system of E. coli. After mining information from various publications, we selected rubisco from Synechococcus elongatus PCC. 7002, which is a well-studied cyanobacteria. Its genome is completely sequenced and it is often used as a model organism for gene manipulation. Previous research has utilized E. coli as a host of random mutagenesis to enhance the activity of Synechococcus rubisco.

How we constructed the design?

Similar to the construction of prk, we codon optimized the sequence of three rubisco subunit and clone it into Psb1c3 plasmid with HindIII and SpeI. The sequence and the size of RbcL is much larger than other subunit, so we separate rbcL from rbcX and rbcS subunits. RbcX and rbcS is separated with a rbsB0034) for the convenience of construction. We attach two different promoters at the upstream of the rubisco. They are P_Lac and PT7 promoter. Since we would like to increase the expression of this protein in the metabolic pathway, we would like to test various promoter combination to find out most efficient combination for our pathway.

How we determine its function?

CA

What is its function?

RuBisCO is the rate-limiting enzyme in carbon fixation. Oxygen competes with CO₂ as a substrate for Rubisco, giving rise to photorespiration. To overcome this problem, some photosynthetic organisms have evolved their own carbon concentrating mechanisms (CCM), which helps to maintain a sufficient amount of CO₂ around RuBisCO.

We are inspired by the carbon concentrating mechanisms (CCM) of cyanobacteria. In cyanobacteria, Rubisco and carbonic anhydrase (CA) is encapsulated in a microcompartment, the carboxysome. Carbonic anhydrase, also known as carbonate dehydratase, is involved in the interconversion between CO₂ and HCO3-. This enzyme can be found in most organisms, including E. coli but the difference is its catalyzing rate in hydration and dehydration of CO₂. Therefore, we will incorporate into our system the carbonic anhydrase gene from Synechococcus elongatus PCC. 7002.

How we constructed the design?

We first codon optimized the sequence and insert it into the pSB1C3 plasmid with HindIII and SpeI just as mentioned above. We link a P_T7 promoter in front of the sequence to enhance the expression. The constructed basic part is then link with other basic parts to complete our construction.

How we tested it function?

The construction of composite part

Rubisco whole protein in pSB1C3

We decided to construct the whole pathway with a dual plasmid system. We link each basic parts with biobrick standard method. We link P_T7-rbcL and P_T7-rbcX-rbcS together. The former, the insert, was digested with EcoRI and SpeI and the later, the backbone, is digested with EcoRI and XbaI. We ligate the backbone with the insert to complete this composite part.

Prk gene into pSB3K3

Prk catalyze the reaction of turning Ru5P into RuBP. Not native to the host, RuBP is, nonetheless, toxic to E. coli. We hope that expression of Prk to be lower in the host so we change the backbone of it into pSB3K3. We selected J04450 from the distributed kit that under the backbone of pSB3K3, which will express red color after the formation of the colony. We digest both backbone and insert with EcoRI and PstI and ligate both fragments. We can then select the colony that does not present in red color if the ligation was conducted successfully.

Link Prk with CA into pSB3K3

We also constructed the composite part that contains both CA and PRK. We construct it using the method mentioned in rubisco whole construction. We cloned the fragments into pSB3K3 for lower expression of prk.

Transformation

After the construction of various composite parts, we co-trasnform them into different E. coli strains mentioned above. Here we listed the strain abbaravatives and its plasmid.

How to prove our design?

pH alert system

The pH alert system, our side project, is a system that allows us to monitor the pH in the surrounding medium in our device at any time by observing the color change of the medium.

The strain that we designed carries an P_asr promoter, a pH-responsive promoter which is native to E. coli and is induced under acidic conditions. In addition, we cloned a sfGFP gene downstream of this promoter whose product will express green fluorescent once the promoter has been activated.

In conclusion, when the color of the medium turns from turbid yellow to green, it indicates the pH of the medium is too low so the medium should be changed as it is not suitable for our E. coli to grow.

Reference