Chlamydomonas and Synechococcus rbcL and CAT coding sequences were amplified separately by PCR, and rbcL-CAT fusions were constructed by overlap extension PCR. A chimeric rbcL-CAT derived from Synechococcus was constructed, but cloning of rbcL-CAT into pENTRTM/D-TOPO was unsuccessful.
Rubisco and Its Limitation
In addition, extensive efforts by researchers to manipulate Rubisco were fueled by the finding that Rubisco carboxylation kinetics vary between species (Esquível, Anwaruzzaman, and Spreitzer 2002). Rubisco kinetics are measured by the ability to distinguish CO2 from O2, known as the specificity factor (Greene, Whitney, and Matsumura 2007).
Functional Screening Method for Rubisco
Identification of Rubisco has attracted more than usual interest as its enzymatic activity is considered to be the rate-limiting step on photosynthesis (Greene, Whitney and Matsumura 2007). Recently, two groups of researchers (Parikh et al 2006; Mueler-Cajar, Morell and Whitney 2007) demonstrated a high-throughput and sensitive screening assay based on heterotrophic Rubisco-dependent E.
Significance and Experimental Design of This Study
The nucleotide sequence of rbcL derived from Chlamydomonas and Synechococcus, CAT genes and GS linker were shown in appendix A, B and C respectively. On the other hand, rbcL derived from eukaryotes cannot be properly folded and may have distorted structures, which can then influence the folding of the CAT downstream.
Objectives of This study
Once this functional selection system is established, sequence swapping between Chlamydomonas and Synechococcus rbcL sequences can be performed, creating libraries of chimeric rbcL-CAT that can be rapidly identified and selected.
Rubisco Enzyme and Agricultural Productivity
The presence of rbcS in form-I Rubisco distinguishes it from the form-II and form-III Rubisco protein complexes, which consist only of rbcL. The simpler form II Rubisco in some cynobacteria and dinoflagellates is a dimer of rbcL subunits, while form III Rubisco, which consists of a pentamer of rbcL dimers, is found in a thermophilic archaeon (Saschenbrecker et al 2007).
Rubisco Catalysis and Limitation
Most studies of Rubisco focus on plant Rubisco which consists of eight rbcL and eight rbcS in a hexadecameric structure (known as Form-I Rubisco). However, in the absence of rbcS, the catalysis of rbcL is significantly reduced and suggested that rbcS has a profound influence on catalysis (Andrews 1988).
Engineering Rubisco to Improve Photosynthetic Efficiency
8 Rubisco carboxylation activity is mainly compromised by its non-specific properties, which are also able to oxygenate RuBP simultaneously with the release of assimilated CO2 (as shown in Figure 2.1). Photosynthesis is the basis of plant growth, and evidence had shown that increased photosynthetic efficiency in crop plants was able to increase yield potential (Evans 2013).
Rubisco Screening Applications
Rubisco Functional Selection Strategies
Functional Screening Method for Rubisco in Escherichia coli
Although these selection methods provide insight into the development of functional screening assays for Rubisco, the limitation of throughput reduces their validity for screening large mutagenic libraries (Mueller-Cajar and Whitney 2008b). Thus, co-expression of PRK and functional Rubisco is crucial to bypass the blockage, especially when cell is provided with minimal medium containing carbon source upstream of glycolytic lesion.
Species Specific Assembly Barrier of Rubisco
Molecular chaperone primarily prevents the aggregation of the polypeptide chain and mediates folding followed by assembly of newly synthesized polypeptides (Saschenbrecker 2007). After GroEL/GroES-mediated folding, RbcX specifically recognizes the C-terminus of RbcL and assists in the formation of stable RbcL8, followed by the displacement of RbcX by RbcS (Saschenbrecker et al 2007).
Recombinant Protein Consisting rbcL and Reporter Gene
After cleavage of protease site by endogenous enzyme, rbcL with short linker peptide at carboxy terminus is found to be able to assemble into functional multimeric holoenzyme and support photoautotrophic growth. This study also suggested that Chlamydomonas rbcL with a short carboxy-terminal linker does not significantly affect its assembly or catalysis.
Degradation of Unstable Protein in Escherichia coli
Summary of Procedure for Molecular Cloning of rbcL
Culture Medium
Two types of culture medium were used in this project, Super Optimal broth with catabolic repressor (S.O.C.) and 2x yeast extract and tryptone (2x YT) medium.
Antibiotic
Solution
Culture media, solutions, glassware, and materials such as pipette tips, toothpicks, PCR tubes, and microcentrifuge tubes were autoclaved at 121 ˚C, 15 psi for 15 min. On the other hand, waste materials and used glassware, including petri dishes and tips, were decontaminated by autoclaving at 121 ˚C, 15 psi for 30 min before washing or washing.
Preparation of Competent Cells
Polymerase Chain Reaction (PCR)
Amplification of rbcL
Amplification of CAT Gene
Overlap Extension PCR for Ligation of rbcL and CAT Gene
PCR reaction mixture and thermocycling conditions for amplification of CAT gene are summarized in Table 3.7 and 3.9. Primer annealing temperature for amplification of (Chl)CAT linker and (Syn)CAT linker was 61.2 ˚C and 66.9 ˚C respectively. Primer annealing temperature for amplification of (Chl)rbcL-CAT and (Syn)rbcL-CAT was 58.2 ˚C and 60 ˚C, respectively.
Colony Screening
The annealing temperature used to fuse both dies together was 63.7 ˚C for (Chl)rbcL-CAT and 65.6 ˚C for (Syn)rbcL-CAT. Colony PCR thermocycling conditions resemble normal PCR, except that ten minutes of initial denaturation at 96 ˚C was required for bacterial cell wall breakage and plasmid release. Primer annealing temperature of all colony PCR was set at 55 ˚C accompanied by 30 seconds of primer annealing time.
Agarose Gel Electrophoresis
In this project, the universal primer F-M13 or R-M13, which binds to the upstream and downstream cloning sites, was used in the screening of recombinant pENTRTM/D-TOPO (see Appendix D). On the other hand, a T7 terminator that binds downstream of the replacement region was used to screen recombinant pDESTTM17 (see Appendix E).
Purification of PCR Product
Transformation
Cloning of rbcL and rbcL-CAT
TOPO Cloning Reaction and LR Cloning
DNA Sequencing
On the other hand, considering the larger size of rbcL-CAT, the recombinant vector carrying (Chl)rbcL-CAT was sequenced with two additional internal primers targeting the insert. Primer F-RbcL-Chl-CAT and R-RbcL-Chl-CAT were used to screen the recombinant vector carrying (Chl)rbcL-CAT.
Competency of Electrocompetent Cells
Transformation efficiency was calculated from the 10-2 dilution of cells (Figure 4.1; . B) because its cell density is within the appropriate colony count range (25-250 CFU) recommended by the U.
Polymerase Chain Reaction (PCR) .1 Amplification of rbcL
Amplification of rbcL-linker
Amplification of CAT-linker
42 4.2.4 Fusion of rbcL-linker with CAT-linker and amplification of the resulting rbcL-CAT chimeric gene. PCR product of (Syn)rbcL-CAT amplification was purified by agarose gel purification to isolate the desired size fragment (Figure 4.8; Lane 3). Concentration and purity of both purified chimeric genes are listed in Table 4.1. Gradient PCR for amplification of (A)(Chl)rbcL-CAT and (B)(Syn)rbcL-CAT were performed by adjusting Ta from 55 ˚C to 60 ˚C using overlap extension PCR.
Colony PCR of Recombinant pENTR TM /D-TOPO .1 rbcL Gene
Chimeric Gene
Both purified chimeric genes were also cloned into pENTRTM/D-TOPO and transformed into electrocompetent Top10 E. 48 Figure 4.11 Colony PCR for positive clone screening consisting of recombinant pENTRTM/D-TOPO carrying (Chl)rbcL- CAT using M13F and R- Cat.
Plasmid Extraction of Recombinant pENTR TM /D-TOPO .1 pENTR-rbcL
Plasmid from positive transformants consisting of pENTR™/D-TOPO carrying (Ch1)rbcL-CAT (CC1, CC6, CC7) were extracted and used as template for insert amplification. PCR products of extracted plasmids with a band size of approximately 2.5 kb were observed in Figure 4.14. Purified recombinant pENTR™/D-TOPO with a positive result in plasmid identification (C8, S20, CC1, CC6, CC7) was sent for sequencing.
Colony PCR of pDEST TM 17 bearing rbcL
Sequence results were aligned with known sequences of the corresponding rbcL gene using A plasmid Editor (ApE) software. DNA sequencing and alignment results showed that clones C8 and S20 had the correct sequences and the cloned genes were inserted in the correct orientation. 52 Figure 4.15 Colony PCR of cells transformed with pDESTTM17 carrying (Chl)rbcL using the T7 terminator and F-RbcL-Chl.
Plasmid Extraction of Recombinant pDEST TM 17
DNA Sequencing of Recombinant pDEST TM 17
Transformation by electroporation
The calculation shown in section 4.1 showed that the competence of the prepared electrocompetent cells is 4.41x107/µg DNA. 57 prepared highly competent competent cells are mainly required for the production of a complete library of a complex genome (Karcher 1995), therefore prepared electrocompetent cells in this study are sufficient for the cloning attempt. Furthermore, there was no sign of cell growth on the negative control (Figure 4.1; .C), indicating that the prepared electrocompetent cells were not contaminated during the preparation process.
Polymerase Chain Reaction
Amplification of rbcL and CAT gene
On the other hand, high Ta ensures high specificity binding of primer, and yet exceeding optimal Ta can cause low product yield due to poor annealing of primers (Rychlik, Spencer, and Rhoads 1990). Accumulation of primer-template during PCR greatly reduces primer-dimer formation, and therefore primer-dimers were observed at higher intensity in the negative control, which contains no template. It is mainly due to non-specific binding of primer set to lower Ta that results in amplification of secondary product which has a size of approx. 4 kb (result not shown).
Overlap Extension PCR
Thus, it is believed that the high background of non-specific products in rbcL-CAT amplification was mainly due to the high GC content of the template used, especially (Syn)rbcL-CAT (53.8% GC). Nonspecific binding in amplification of (Syn)rbcL-CAT (2.1 kb) resulted in amplification of secondary products that were approximately 650 bp and 1.6 kb. The purified PCR product from the amplification of (Chl)rbcL-CAT and (Syn)rbcL-CAT was found to be pure and consist only of the desired overlap extension PCR product based on agarose gel electrophoresis (Figure 4.8).
Colony Screening of Recombinant pENTR TM /D-TOPO
Troubleshooting Cloning of Chimeric Synechococcus rbcL-CAT PCR-amplification of (Syn)rbcL-CAT resulted in multiple non-specific products
It is suggested that modified low melting point agarose gel offers better alternatives for the purification of DNA. It is suggested that more optimization steps, especially the temperature of the primer hybrid, should be performed to screen the desired gene under optimal conditions. Thus, it is postulated that a reduction in the elongation temperature may increase the efficiency of colony PCR.
Verification of Recombinant pENTR TM /D-TOPO
Restriction digestion of extracted plasmids and screening of desired plasmids by evaluation of their digested size can act as a classic yet reliable additional confirmation test. However, since RNA contamination can lead to serious consequences, including overestimation of plasmid concentration and interference with sequencing reaction, it is suggested that precaution should be implemented. Apart from measuring A260/A280 purity ratio, agarose gel electrophoresis is a simple method that reviews the yield and purity of purified plasmid where a faint and smearing band below the plasmid DNA band will be observed if RNA contamination is present.
DNA Sequencing of Recombinant pENTR TM /D-TOPO
69 chromatogram of CC1 and CC6 had an area with low signal intensity and high background noise. Since the respective gap was only covered by a single read, the identity of CC1 and CC6 was questionable. It is suggested that the 'dirty' sequence was mainly due to RNA contamination, as A260/A280 of both plasmids exceeded 2.0.
LR Recombination of Recombinant pENTR-rbcL with pDEST TM 17 Incorporated rbcL derived from Chlamydomonas and Synechococcus in
This project focused on the construction and cloning of rbcL-CAT derived from Chlamydomonas and Synechococcus. Upon successful cloning of (Syn)rbcL-CAT into pENTRTM-D/TOPO, both Chlamydomonas and Synechococcus pENTR-rbcL-CAT will be subcloned into expression vector, pDESTTM17. Cloning of the gene for phosphoribulokinase activity of Rhodobacter sphaeroides and its expression in Escherichia coli.
Directed evolution of Rubisco in Escherichia coli reveals a specificity-determining hydrogen bond in the form II enzyme. Sequence alignment of the expected (Syn)rbcL-CAT sequence and the (Syn)rbcL-CAT PCR amplification product.
