The photosynthetic bioreactor research program is a training platform appropriate for introducing advanced molecular biology techniques to undergraduate students and advanced high school biology students. For this advanced molecular biology training exercise, the enzyme carbonic anhydrase was cloned, over-expressed, purified, and functionally characterized. Carbonic anhydrases are industrially important enzymes with potential use in carbon sequestration and biofuel production. Alpha and beta carbonic anhydrases from Photobacterium profundum, a psychrophilic, halotolerant bacterium, were characterized in this study. Carbonic anhydrases that can withstand high salinity and are active at low temperatures can be transformed into oleaginous marine microalgae to enhance biofuel production. Our research program started with a three-day boot camp with lectures in relevant topics of molecular biology, microbiology, and research methods. After the boot camp, the lab phase of the project involved training students to perform polymerase chain reaction, DNA gel electrophoresis, DNA ligation, and bacterial transformation. In the final phase of the project, students were trained in recombinant protein over-expression and protein purification techniques. Here we report successful cloning and over-expression by high school students of two novel carbonic anhydrases from a psychrohalophile with application in biofuel production.
Carbonic anhydrases (CA) are metalloenzymes that are important for critical physiological functions in living organisms. In plants, carbonic anhydrases catalyze interconversion of carbon dioxide (CO2) to bicarbonate, which is utilized by ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) during photosynthesis (Badger & Price, 1994). It has been suggested that functional transformation of plants with prokaryotic carboxysomes containing carbonic anhydrase enhances photosynthesis, which in turn leads to increased biomass production (Price et al., 2013). Oleaginous microalgae have been proposed as a viable alternative to fossil fuels, but the cost of production and extraction of lipids from microalgae remains very high. Microalgae such as Dunaliella and Nannochloropsis thrive in hypersaline (high-salinity) environments and have optimal growth temperatures ranging from 25 to 30°C. Bioengineering microalgae with carbonic anhydrases isolated from extremophiles that live in similar conditions should ideally result in functional enzymes, since proteins from extremophiles that live in vastly different conditions may result in proteins that are not functional. Furthermore, over-expressing carbonic anhydrase in microalgae has been shown to enhance growth (Kikutani et al., 2016), and engineered microalgae with carbonic anhydrases that have higher activity than native enzymes should result in increased growth, which ultimately results in higher lipid production as a function of greater microalgal biomass per unit time.
Bioengineering microalgae with enhanced growth and lipid production using carbonic anhydrases from extremophiles can circumvent the costs associated with microalgal biofuel production. CAs are a well-studied type of enzyme and have been classified into six different groups (ɑ, β, γ, δ, ζ, and η) (Di Fiore et al., 2015). Of these six groups, ɑ, β, and γ CAs have been extensively characterized (Huang et al., 1998; Reed & Graham, 1981; Fukuzawa et al., 1992; Ferry, 2010); the delta and zeta carbonic anhydrases (δ-CA and ζ-CA) are found only in algae and diatoms; and the sixth group, η-CA, was recently identified in an important pathogenic protozoan, Plasmodium falciparum (Del Prete et al., 2014). Even though CAs are very well studied, most of the work has been carried out on mesophilic and thermophilic CAs. To date, very few psychrophilic CAs have been characterized.
Biochemical and structural characterization of any enzyme is dependent on its successful over-expression and solubility. E. coli-based over-expression of a recombinant protein is a basic molecular biology procedure that is used in research laboratories as well as biotechnology industries. Even though today's high school students learn about some basic molecular biology techniques such as DNA electrophoresis, bacterial transformation, or polymerase chain reaction (PCR), they rarely get an opportunity to learn how these techniques are used in discovery research.
Our National Science Foundation funded project, Emerging Frontiers in Research and Innovation—Photosynthetic Biorefinery (EFRI-PSBR), is a multidisciplinary effort aimed at making algal biofuels an economically viable option to fossil fuels. The Photosynthetic Biorefinery Summer High School Research program (PBSHR) is a part of the EFRI-PSBR project that is dedicated to training high school students in the central dogma of molecular biology, discovery research methods, and molecular biology techniques, and can be used to provide advanced molecular biology training in undergraduate biology laboratories as well. We selected two uncharacterized carbonic anhydrases from Photobacterium profundum to train the high school students in cloning and in recombinant protein expression. P. profundum is a psychrohalopilic barophile first isolated from deep sea sediment. P. profundum has an optimum growth temperate of 15°C and can withstand pressures between 0.1 MPa to 90 MPa (Vezzi et al., 2005). The students were able to successfully amplify gene encoding for an ɑ-CA and β–CA from genomic DNA, which were subsequently cloned into an expression vector. Furthermore, the students over-expressed the carbonic anhydrases in an E. coli-based expression system and were able to show that the over-expressed proteins were soluble. Enzyme assays using a novel technique known as protonography showed that both these enzymes exhibited CO2 hydration activity. This is the first report describing the successful purification and enzymatic activity analysis of psychrophilic carbonic anhydrases from P. profundum.
Materials & Protocols
Bacterial Strains, Growth Conditions, and Chemicals
E. coli cells were routinely grown in Luria-Bertani (LB) media at 37°C with shaking at 200 rpm, and supplemented with 35 μg/ml kanamycin for plasmid maintenance. E. coli XL1 Blue cells were obtained from Stratagene, and the expression host, E. coli BL21(DE3) was obtained from Novagen. P. profundum genomic DNA was purchased from the American type culture collection (ATCC). PCR was performed using a Bio-Rad C1000 TouchTM thermal cycler (Bio-Rad), and the iProofTM hi-fidelity polymerase used in PCR reactions was purchased from Bio-Rad. NdeI and XhoI restriction enzymes were purchased from New England Biolabs. Over-expression vector, pET28a, was obtained from Novagen. Isopropyl-β-thiogalactoside (IPTG) used for protein induction was purchased from Fisher Scientific, and the protein purification was carried out using Ni-NTA (nickel-nitrilotriacetic acid) spin columns (Qiagen).
PCR Amplification and Cloning of P. profundum ɑ–CA and β–CA
PCR amplification of ɑ–CA and β–CA genes was carried out using P. profundum genomic DNA as a template. Gene specific primers were designed previously by the research fellow and synthesized by Eurofins MWG operon. The gene encoding for ɑ–CA was amplified using primers Ppr_alpha P1 (5′-GGA ATT C CAT ATG GCT GAA TGG AGT TAT ACT GGC G-3′) and Ppr_alpha P2 (5′-CCG CTC GAG TTA TTC TAA GAT CAG GCG CGC-3′) (restriction enzyme sites are underlined). β-CA was amplified using primers Ppr_beta P1 (5′-GGA ATT C CAT ATG ATG GCA GAT ATT AAG CAG TTA TTC G-3′) and Ppr_beta P2 (5′-CCG CTC GAG CTA CAA TTC TTT TGG TGG TAG AAT TG-3′) (restriction enzyme sites are underlined). The PCR reaction mix and cycle sequence used is detailed in the Supplementary Material section. The resulting 657 bp product for ɑ–CA and 675 bp product for β–CA were gel-extracted using the QIAquick Gel Extraction Kit (Qiagen) following the manufacturer's instructions. The gel-extracted PCR products were digested overnight at 37°C using NdeI and XhoI restriction enzymes. After restriction digest, the PCR products were gel-extracted and ligated with pET28a vector that was digested with the same enzymes and purified using gel-extraction. Ligation reactions were carried out using T4 DNA ligase (New England Biolabs) at 16°C overnight. The ligations were then mixed with calcium-chloride-competent E. coli XL Blue cells, and transformations were carried out using the heat-shock method (Oishi & Cosloy, 1972). Ligated plasmids were purified from the transformants using a Qiagen plasmid purification kit following the manufacturer's instructions, and the presence of CA genes in the plasmids was confirmed by whole cell PCR (see Supplementary Material) and gene sequencing using commercially available primers, T7 (5′-TAA TAC GAC TCA CTA TAG GG-3′) and T7 terminator (5′-GCT AGT TAT TGC TCA GCG G) (Eurofins MWG).
Over-Expression of 6xHistidine-Tagged Carbonic Anhydrases from P. profundum
Over-expression of the CAs was performed by transforming the recombinant plasmid containing ɑ-CA or β–CA into the E. coli BL21(DE3) (Novagen) expression strain, and the transformants were then inoculated in Luria-Bertani (LB) broth containing kanamycin (35 μg/ml) and grown overnight at 37°C with shaking (200 rpm). The E. coli BL21(DE3) strain supports over-expression of protein from plasmids with genes driven by T7 promoter sequences. T7 promoters are recognized with high affinity by the T7 bacteriophage encoded RNA polymerase, which supports strong expression of genes that are driven by the T7 promoter (Studier et al., 1990). The E. coli BL21(DE3) strain is particularly useful for T7-based overexpression because the strain has the T7 RNA polymerase encoded in its genome (the DE3 sequence) under the control of the IPTG-inducible lac promoter such that when IPTG is added to the BL21(DE3) cultures, the T7 RNA polymerase is produced, which then enables high-level expression of the T7 promoter-driven target gene coded for in the expression plasmid. The overnight cultures were used to inoculate 250-ml flasks containing 50 ml of LB media and supplemented with 35 μg/ml of kanamycin (5% inoculum), and the cultures were incubated at 37°C with shaking (200 rpm), until an optical density of 0.6 (600 nm) was reached for each culture. The culture growth was monitored using a Biorad SmartSpec 3000 (Bio-Rad). The cultures were then incubated at 20°C with shaking (200 rpm), and protein expression was induced with 0.5 mM IPTG. After overnight incubation, the cells were harvested by centrifugation (4000 rpm, 20 min at 4°C), and the cell pellets were stored at −20°C until use.
Solubility Analysis of Over-Expressed ɑ–CA and β–CA Proteins
Solubility of both the CAs was tested by thawing the frozen pellets in an ice-bucket filled with ice, followed by resuspending the pellets in 20 mM Tris pH 7.5 and 150 mM NaCl. The resuspended pellets were lysed with B-PERTM bacterial protein extraction reagent (Thermo Scientific) with vigorous shaking for 30 minutes at room temperature. An aliquot of lysed cells was set aside as total protein, and the remaining lysate was centrifuged for 30 minutes at 13,500 rpm at room temperature. The supernatant containing soluble proteins was transferred to a new Eppendorf tube, and the remaining pellet containing insoluble proteins was resuspended in 20 mM Tris pH 7.5 and 150 mM NaCl. The total, soluble, and insoluble protein fractions were mixed with Laemmli loading buffer (Laemmli, 1970), and the proteins were separated on a 12.5 percent polyacrylamide gel. The separated proteins were visualized by staining the gel in coomassie brilliant blue dye.
Purification of Over-expressed ɑ–CA and β–CA
Frozen cell pellets were thawed in ice and resuspended in Buffer A (20 mM Tris pH 7.5, 500 mM NaCl, 30 mM imidazole, 1 mM benzamidine, and 1 mM phenyl-methylsulfonly fluoride [PMSF]). Cells were lysed with B-PERTM bacterial protein extraction reagent with vigorous shaking for 30 minutes at room temperature. The lysed cells were centrifuged at 12,000 × g for 30 minutes at room temperature to remove the cell debris. Small-scale purification of ɑ–CA and β–CA was done using Qiagen Ni-NTA spin columns following manufacturer's instructions. The spin columns were equilibrated with binding buffer containing 20 mM Tris pH 7.5, 150 mM NaCl, and 30 mM Imidazole by centrifuging for 2 minutes at 890 × g. After equilibration, cleared cell lysate containing histidine-tagged carbonic anhydrase was loaded on to the spin columns and centrifuged for at 270 × g for 5 minutes to facilitate binding. The spin columns were washed twice by centrifuging at 890 × g for 2 minutes with wash buffer containing 20 mM Tris pH 7.5, 150 mM NaCl, and 60 mM Imidazole to remove weakly bound proteins. Bound histidine-tagged proteins were eluted by centrifugation at 890 × g for 2 minutes using buffer containing 20 mM Tris pH 7.5, 150 mM NaCl, and 500 mM or 1 M imidazole. The presence of proteins in the elutions was confirmed by SDS-PAGE. Purified proteins were pooled and dialyzed overnight at 4°C in dialysis buffer (20 mM Tris pH 7.5, 150 mM NaCl) using a Slide-A-lyzer 10K dialysis cassette (Thermo Scientific).
“In-gel” Carbonic Anhydrase Assay
In-gel carbonic anhydrase activity assays, otherwise known as protonography, were carried out as described by De Luca et al. (2015). Purified carbonic anhydrases were mixed with Laemmeli loading buffer without 2-mercaptoethanol and loaded on a 12.5 percent SDS-PAGE. The gel was run at 150 V for approximately 45 minutes. Visualization of carbonic anhydrase activity was done by soaking the gel in 2.5 percent Triton X-100 for 1 hour on a shaker and washed twice in a buffer containing 100 mM Tris pH 8.2 and 10 percent isopropanol for 10 minutes. The gel was then incubated in 0.1 percent bromothymol blue in 100 mM Tris pH 8.2. Finally, the gel was immersed in CO2-saturated water to visualize carbonic anhydrase activity. CO2-saturated water was prepared by bubbling CO2 gas for 3 hours in 250 ml of ddH2O at room temperature.
Student Learning Objectives
After the completion of the PSBHR program, the students should be able to:
Have a working knowledge of basic molecular biology laboratory techniques such as electrophoresis, PCR, recombinant protein expression, and protein purification, which will enable the students to conduct the techniques independently. It is also expected that the students will be able to explain clearly the underlying molecular biology concepts that enable understanding of how the molecular biology techniques work as well as how the techniques are performed.
Deliver clear and accurate scientific presentations.
Compile and organize scientific data in lab notebooks.
Use compiled data to design posters for scientific presentations.
Results, Outcomes, and Discussion
The six-week PBSHR research project for the students (rising high school juniors) began with an intensive three-day boot camp. The students were trained by graduate students and a research fellow in basic microbiological techniques, media preparation, bacterial and algal growth monitoring, and pipetting techniques. In addition to laboratory techniques, the students attended lectures delivered by faculty members, graduate students, and research fellows on various topics such as basic molecular biology, microbiology, and basic algal physiology. Specific topics on gene cloning, restriction enzymes, electrophoresis, chromatography, and PCR were also covered during the boot camp. The three-day intensive boot camp provided the students with the background required to work in a research-intensive environment. After the boot camp, the students started their assigned projects with continuous monitoring and help from the mentors.
Weeks 1 and 2: PCR Amplification and Cloning of Target Genes into the Expression Vector
The students were provided with instructions for performing PCR to amplify the genes encoding ɑ-CA and β-CA from P. profundum genomic DNA. Prior to the start of the cloning and over-expression of carbonic anhydrases, every step of the process was explained in detail to the students. Subsequently, with the help of the research fellow, the students were tasked with designing a schematic of the entire cloning and over-expression process to aid the students in understanding the underlying principles involved in cloning and recombinant protein expression as well as the project workflow (Figure 1).
The primers used for the amplification of the genes were designed previously by the research fellow. Following the PCR protocol and instructions provided to them, the students were able to successfully amplify the CA genes from P. profundum. PCR amplification using gene-specific primers resulted in 657 bp product for ɑ-CA and 675 bp product for β-CA (Figure 2). The resulting gene products were purified by gel-extraction. The students were provided with manufacturer's protocol for gel-extraction and were given time to read the instructions first. Subsequently, the students, with guidance from research fellow, successfully gel-extracted the PCR product. The digested PCR products were ligated with a pET28a vector that was previously digested with the same restriction enzymes that were used to digest the PCR products. Ligations were carried out with a commonly used, bacteriophage-derived ligase known as T4 DNA ligase. The students were successfully able to use T4 DNA ligase to produce recombinant pET28a plasmid containing either ɑ–CA or β–CA. The presence of the inserts was confirmed using whole-cell PCR and sequencing.
Week 3 and 4: Carbonic Anhydrase Expression and Solubility
Successful expression of recombinant protein is the foremost and essential step in elucidating the function for any protein. A wide variety of expression vectors are commercially available for this purpose, each with its own unique advantages and disadvantages. One of the most commonly used vectors for the heterologous expression of a target protein is based on a strong T7 bacteriophage promoter. The use of T7 promoters for the expression of recombinant proteins was first described by Studier et al. in 1990. Commercially available pET vectors that enable cloning of genes downstream of the T7 promoter are widely used in biotechnology industries as well as in research laboratories for protein expression. SDS-PAGE analysis of over-expressed ɑ–CA and β–CA revealed the presence of 35 kDa bands and 25 kDa bands, respectively (Figure 3). Furthermore, analysis of total, soluble, and insoluble fractions revealed that both the carbonic anhydrases are soluble under the conditions tested (Figure 3).
Week 5: Carbonic Anhydrase Purification Using Metal Affinity Chromatography
Both ɑ–CA and β–CA were expressed with an N-terminal 6xHistidine tag to facilitate purification using affinity chromatography. Prior to the start of the purification process, the principle behind affinity chromatography was discussed with the students. Affinity tags are peptide sequences that are generally attached to the N- or C-terminus of a recombinant protein to facilitate purification. Histidines are amino acids that have high affinity to nickel ions. This property has been exploited extensively in the purification of recombinant proteins that are tagged with N- or C-terminal histidine tags. Most of the commercially available affinity chromatography columns use NTA as the adsorbent to immobilize nickel ions, and the use of Ni-NTA for protein purification was first demonstrated by Hochuli et al. (1987).
Binding of the target protein to the Ni-NTA resin was achieved by passing the lysate-containing, histidine-tagged protein through the Ni-NTA column. Elution of bound protein is achieved either by changing the pH of the column buffer or by using a linear gradient of imidazole. Purification of histidine-tagged ɑ–CA and β–CA was done using Qiagen Ni-NTA spin columns. The students were given the manufacturer's protocol and were expected to read the protocol, understand it, and execute the purification process following the instructions. The students were closely supervised by the research fellow during the purification process. The Ni-NTA spin columns are relatively inexpensive and are an easy method of purifying histidine-tagged recombinant proteins. The protocol calls for several simple centrifugation steps and can be easily done by anyone, even with little or no training in protein purification. Both the students completed the purification process, and the success of their purification was analyzed by SDS-PAGE. The presence of distinct 35 kDa bands for ɑ-CA and 25 kDa bands for β–CA in the elution fractions indicate that both the proteins were successfully purified using Ni-NTA spin columns (Figure 4). The purified proteins were pooled, and dialyzed against 20 mM Tris pH 7.5, 150 mM NaCl at 4°C overnight to remove imidazole. The purified carbonic anhydrases were stored at −80°C until further use.
Week 6: Carbonic Anhydrase Activity Assay
Carbonic anhydrases are enzymes that catalyze the inter-conversion of carbon dioxide and water to bicarbonate and a proton (Tripp et al., 2001). Activity assays for carbonic anhydrases generally relied on the rapid change in the pH of a buffer from 8.2 to 6.3, and this change is monitored using a pH meter or colorimetrically using a pH indicator. Even though these methods are well established and routinely used for carbonic anhydrase assays, the complexity of the process makes it difficult for students with very little background in enzymology to understand and interpret the data obtained from these assays. In-gel activity assays are simple, cheap, and an easy alternative to complex activity assays. Recently, a novel in-gel activity assay for carbonic anhydrases known as protonography was developed by De Luca et al. (2015).
This assay relies on the reversible inhibition of carbonic anhydrase activity by SDS. Carbonic anhydrase separated by SDS-PAGE can be reactivated through removing the SDS by treating the gels in an aqueous solution of Triton X-100. The gel is then stained with bromothymol blue, a pH indicator that changes color from blue to yellow in response to pH change from 8.2 to 6.8. Carbonic anhydrase activity is detected by immersing the bromothymol blue stained gel in CO2-saturated water; the appearance of yellow bands indicates the local reduction of pH due to carbonic anhydrase activity (Tripp et al., 2001). Both ɑ–CA and β–CA were separated on a SDS-PAGE, and protonography analysis revealed the appearance of yellow bands, indicating that both these proteins exhibit carbonic anhydrase activity (Figure 5).
Conclusions and Evidence of Student Learning
SDS-PAGE analysis revealed that both ɑ–CA and β–CA were successfully over-expressed, and both the proteins were soluble under the conditions tested. As a part of the EFRI-PSBR project, the crystal structure of the ɑ–CA purified by one of the high school students has been solved (Somalinga et al., 2016), and the preliminary biochemical analysis showed both ɑ–CA and β–CA carbonic anhydrases are catalytically active. Both the CAs will be further characterized and subsequently used for bioengineering microalgae to enhance biofuel production. The PBSHR is an ongoing project and has gained immense interest among the students at Research Triangle High School. The students that participate in the program receive invaluable experience in advanced molecular biology and research techniques. In addition to the research experience, the students were also trained in designing scientific posters (Supplementary Figures S1 and S2) and delivering scientific talks in a professional scientific forum. Furthermore, the PBSHR program is designed to be implemented in any undergraduate or high school biology teaching laboratory with little or no modification. Every step of the process has been standardized to ensure reproducibility, and any teaching laboratory planning to run the PBSHR module will be provided with necessary primers, plasmids, or expression strains.
At the end of the program, questionnaires were provided to each student to evaluate the effectiveness of the PBSHR program (Supplementary Table S1). The PBSHR program not only trains high school students in discovery research but also helps in generating important data that will be used by senior researchers in advancing fundamental biological sciences research, with the high school students serving as contributing authors (Somalinga et al., 2016).
This work was supported by the National Science Foundation EFRI program under Grant Number EFRI 1332341.