A Step-by-Step Approach to Establishing an Efficient Genetic Transformation Protocol for Chlorella vulgaris Using Electroporation

A Step-by-Step Approach to Establishing an Efficient Genetic Transformation Protocol for Chlorella vulgaris Using Electroporation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Method Article A Step-by-Step Approach to Establishing an Efficient Genetic Transformation Protocol for Chlorella vulgaris Using Electroporation Dexter Miller Robben, Zarina Amin, Cahyo Budiman, Vijay Subbiah Kumar This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7100769/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Oct, 2025 Read the published version in Molecular Biology Reports → Version 1 posted 11 You are reading this latest preprint version Abstract Chlorella vulgaris , a unicellular green microalgae, has shown diverse applications in biotechnology. Particularly in microalgae genetic engineering, genetic transformation of C. vulgaris offers a sustainable and cost-effective approach to producing recombinant protein. Despite this, the thick cell wall of C. vulgaris poses a challenge, often necessitating optimization of the transformation parameters to overcome the challenge. This current study presents a detailed methodology for efficient electroporation-mediated transformation of C. vulgaris . The electroporation procedure described here includes C. vulgaris cell preparation and electroporation parameters such as pulse voltage, capacitance, and resistance. Prior to the cultivation of the transformed cells in a selective BG11 medium, recovery conditions of the newly transformed cells in a regular BG11 medium were also described. Through optimization of these parameters, an improved transformation efficiency while maintaining cell viability was achieved. Positive amplifications of the pCambia1303 t-DNA region in the PCR and RT-PCR assays were indicative of the success of the electroporation-mediated transformation of C. vulgaris . DNA sequencing was then used to confirm the identity of these amplicons, thus validating the successful transformation procedure. The electroporation-mediated transformation procedure established here provides a reliable and reproducible method for carrying out genetic modification on C. vulgaris to facilitate its potential use as a bio-factory in numerous biotechnological applications. microalgae genetic engineering electroporation recombinant DNA Figures Figure 1 Figure 2 Introduction Microalgae are known for their diverse biotechnological applications, ranging from biofuel production to pharmaceuticals [ 1 ], [ 2 ], [ 3 ]. Additionally, the emergence of microalgae genetic engineering has provided a promising avenue for an alternative recombinant protein production system that is favourable over the traditional bacterial and yeast expression systems [ 4 ], [ 5 ], [ 6 ]. C. vulgaris , a green microalga belonging to the Chlorophyta family, has shown potential as a host organism for genetic transformation. The reason for this is due to its single-celled structure, which allows for a simple genetic transformation technique to be carried out. Additionally, the minimal cultivation requirements and rapid growth rate have allowed for the development of an alternative expression system with high scalability, while at the same time aligning with sustainable biotechnological practices. One of the most widely used transformation methods is electroporation. It facilitates the insertion of foreign genetic material into cells across diverse taxa, ranging from prokaryotes to eukaryotes. This method of transformation utilizes electrical pulses to create pores through the membrane of the cell, allowing an opening for foreign DNA to enter the cell. Consequently, the foreign DNA is incorporated into the genome of the host cell [ 7 ], [ 8 ], [ 9 ], [ 10 ], [ 11 ]. In Chlorella sp. , it is essential to establish an efficient genetic transformation method to further advance its applications in fundamental research in algal biotechnology. These transgenic lines of C. vulgaris may play a significant role in the current biotechnological applications through their enhanced and improved yield and nutritional value, or their capability as a biological factory to produce a desired protein [ 7 ], [ 10 ], [ 12 ], [ 13 ]. Notwithstanding, the recalcitrant nature of the C. vulgaris cell wall poses a major challenge to a successful transformation. Due to its thick cell wall (15–20 nm), C. vulgaris is often treated with a mixture of enzymes to generate protoplasts prior to the transformation procedure [ 7 ], [ 14 ], [ 15 ]. This limitation has hampered progress in genetic engineering efforts for C. vulgaris. As a result, the development of an efficient transformation method remains vital for advancing research involving this species. This methodology paper details the procedure for efficient electroporation-mediated transformation of C. vulgaris. Optimized electroporation parameters, recovery conditions, and the selection of transformants were highlighted. The described electroporation procedure provides a streamlined and effective approach for generating a transgenic line of C. vulgaris strains, providing a robust tool for researchers to further study the biotechnological potential of this green microalga. Materials and Methods C. vulgaris culture was obtained from Algae Research Supply (Carlsbad, California), and all procedures related to the transformation of recombinant DNA and the cultivation of the transgenic C. vulgaris were performed in a containment facility (Transgenic facility of Biotechnology Research Institute, Universiti Malaysia Sabah). Figure 1 shows an overview of the entire electroporation-mediated transformation workflow. Step-by-step protocol Step 1: Cultivation of C. vulgaris Cultivate C. vulgaris in BG11 medium (pH 6.8) at an ambient temperature of 28°C under a 16:8 photoperiod in a growth chamber. Subculture 10 mL of C. vulgaris mother culture to 40 mL of BG11 medium under the same conditions as in the previous step. Use C. vulgaris cells in the early growth phase of 7 days after subculture for the transformation by electroporation procedure. Comments and troubleshooting In this study, the BG11 medium was used for the cultivation of C. vulgaris . It is important to monitor the pH of the BG11 medium regularly, as deviations from the optimal range of approximately pH 6.8 can impact the culture growth. In addition, temperature should be held consistently at 28°C, and the light regime follows a 16:8 photoperiod. We recommend manually agitate the culture once a day by gently swirling if a shaker is not available, as this will facilitate the dispersed cells to be uniformly exposed to light and nutrients. When preparing the culture for electroporation, it is important to subculture the cells from the mother culture into fresh BG11 medium and allow them to grow for 7 days. This 7-day-old subculture, rather than the mother culture itself, should be used for the electroporation procedure. This is because cells in the early growth phase are more likely to have thinner cell walls and higher metabolic activity, making them more amenable to transformation. Overgrown cultures that have entered the stationary phase often have thicker cell walls, which can reduce transformation efficiency [ 7 ], [ 16 ], [ 17 ]. To prevent contamination, it is crucial to practice aseptic techniques. If contamination arises, the affected culture should be discarded, and a new culture should be initiated. Step 2: Preparation of C. vulgaris cells Harvest cells (1×10 7 − 1×10 8 cells per mL) through centrifugation at 10,000 rpm for 2 min at 4°C. Wash the pelleted cells with 1 mL of 384 mM D-sorbitol at 2,500 rpm for 5 min at 4°C. Repeat the washing step three more times. Comments and troubleshooting When preparing C. vulgaris cells, ensure that the cell density is within the range of 1×10⁷ – 1×10⁸ cells per mL before harvesting [ 12 ]. Too few cells may result in low transformation efficiency, while too many cells can lead to clumping and uneven washing. Follow the specified speed and conditions of the centrifugation to avoid cell damage. The higher speed (10,000 rpm) during harvesting ensures efficient pelleting, while the lower speed (2,500 rpm) during washing helps maintain cell integrity. Washing steps with 384 mM D-sorbitol are critical to remove residual salts, which can interfere with electroporation [ 18 ]. In this study, we recommend that the washing of the cells be done 4 times to ensure a thorough washing of the cells for an efficient transformation by electroporation. Maintaining sterile conditions throughout the procedure is also essential to avoid contamination. Use sterile tubes, pipettes, and solutions, and work in a clean environment to ensure. Step 3: Transformation by electroporation technique Add 1 mL of 384 mM D-sorbitol to the washed cells and resuspend by pipetting up and down. Add 1 µg of pCambia1303 vector and mix by gently flicking the tube. Transfer the mixture to an electroporation cuvette and incubate the mixture on ice for 10 min. Place the cuvette in the electroporation pod and subject it to a single-pulse electroporation with an electric field of 2.2 kV, a capacitance of 50 µF, and a resistance of 500 Ω using a Bio-Rad Gene Pulser II (Biorad, USA). Place the cuvette and incubate the mixture on ice for 10 min. Transfer electroporated cells to a 15 mL Falcon tube containing 10 mL of BG11 medium and incubate the cells at room temperature for two days in low light exposure. Comments and troubleshooting It is crucial to ensure that the cells to be transformed are resuspended evenly in 384 mM D-sorbitol. Resuspension should be done by gently pipetting up and down until a homogenous mixture is observed. The use of a high quantity of plasmid DNA is also important to ensure a high possibility of the DNA being taken up by the C. vulgaris cells. In this study, we use the pCambia1303 vector at a concentration of 1 µg. Cooling of cells before and after electroporation is essential to maintain the permeability of the cell membrane and improve the DNA uptake. Skipping this step or insufficient cooling can lead to poor transformation results. We recommend an incubation period of the C. vulgaris cells (before and after electroporation) on ice for 10 min. In this study, the electroporation parameters used are 2.2 kV, 50 µF, 500 Ω. These parameters are effective for the transformation of C. vulgaris without prior enzyme treatment. Due to the thick cell wall of C. vulgaris , a high voltage is employed. Arcing is a common occurrence in electroporation procedures that is usually caused by the presence of salts. Thus, it is important to ensure that the cells are sufficiently washed to remove residual salts. If transformation efficiency is low, consider optimizing the parameters for your specific setup. Following the electroporation of the C. vulgaris cells and an incubation period on ice, immediately transfer the cells to BG11 medium to minimize stress and allow for cell recovery [ 7 ], [ 19 ]. This step is known as the resting period post-transformation by electroporation. It is recommended that the incubation period be two days at room temperature under low light. If cell viability is low after electroporation, try reducing the electric field strength slightly or increasing the recovery time. Step 4: Cultivation of transformed C. vulgaris in selective medium Following the 2-day resting period, subculture 100 µL of the transformed C. vulgaris culture to a 5 mL BG11 medium supplemented with 50 µg/mL of hygromycin B. Maintain the culture conditions at an ambient temperature of 28°C under a 16:8 photoperiod in a growth chamber. Observe for growth daily. Growth is expected to be visibly observed after seven days of cultivation. Comments and troubleshooting It is important to use the correct concentration of hygromycin B (50 µg/mL) when cultivating the transformed C. vulgaris . This is to ensure effective selection of the transformed cells. Avoid fluctuations of the culture conditions, such as temperature and light exposure, as these may cause stress to the cell and delay growth. If no growth is observed after 7 days, ensure that the antibiotic used has not expired and verify its concentration. Discard the culture if contamination is observed and restart the culture with sterile techniques. Lastly, increase the initial inoculum volume slightly or extend the growth period if growth is slower than expected. Some transformed cells may take longer to adapt to the selective medium. A lower concentration of antibiotic (i.e., 30 µg/mL) can be used at the initial subculture before using a higher concentration of antibiotic at the subsequent culture. Regular monitoring and adjustments will help ensure the successful cultivation of transformed C. vulgaris . Step 5: Genomic DNA and total RNA isolation from the transformed C. vulgaris cells Harvest the transformed C. vulgaris cells by centrifugation at rpm for min at 4°C. Extract the gDNA and total RNA of the harvested cells using the Wizard® Genomic DNA Purification Kit (Promega, USA) and PureLinkTM RNA Mini Kit (Thermo Fisher Scientific, USA) following the manufacturer’s protocol, respectively. Electrophorese the isolated gDNA and total RNA in 1% agarose gel. Perform electrophoresis at 70 V for 30 min. Stain the gel in ethidium bromide solution for 10 min. View the gel under the Gel Doc EZ Imager (Bio-Rad, USA). Comments and troubleshooting The use of other commercially available kits for the gDNA and RNA extractions is feasible. However, it is important to ensure the compatibility of the kit to the type of sample under study. In this study, the Wizard® Genomic DNA Purification Kit (Promega, USA) and PureLinkTM RNA Mini Kit (Thermo Fisher Scientific, USA) were used for the isolation of the gDNA and RNA from wild-type and transformed C. vulgaris cells. When harvesting the C. vulgaris cells, ensure cells are thoroughly centrifuged at the specified speed and time (10,000 rpm for 10 min at 4°C). The starting volume of the culture may be increased if the pellet obtained after centrifugation is too small. Follow the manufacturer’s instructions for the kits used for the isolation of nucleic acids to ensure a good-quality yield. Especially when isolating RNA, degradation can occur if proper precautions are not taken, such as using RNase-free consumables and working in an RNase-free environment. For resolving gDNA and RNA samples, we recommend using a 1% agarose gel to separate the nucleic acids effectively and perform the gel electrophoresis at 70 V for 30 min. Following the completion of the gel electrophoresis, stain the gel appropriately, ensuring safety precautions are followed if staining is done with ethidium bromide. Ensure that the intact bands of DNA and RNA are present before proceeding to the next step. Step 6: PCR and RT-PCR assay targeting the pCambia1303 vector intergenic region (~ 120 bp) For the RNA sample, perform cDNA synthesis using the SuperScript™ IV First-Strand Synthesis System (Thermo Fisher Scientific, USA) following the manufacturer’s protocol. Prepare a 20 µl PCR mix with the following reagents to generate amplified copies of the gDNA and cDNAs: 1 µl of gDNA or cDNAs, 1 µl of Taq polymerase (5 U/µl, Promega, USA), 4 µl of 5× PCR buffer, 0.4 µl dNTPs (10 mM), 1.2 µl MgCl2 (1.5 mM), 0.6 µl of each PCR primer (25 µM), and top up the volume with nuclease-free water. The amplification program is set for an initial denaturation at 95°C for 5 min, with 35 cycles of denaturation at 95°C for 45 s, annealing at 55°C for 30 s, and extension at 72°C for 45 s, followed by final extension at 72°C for 5 min. Analyse the PCR amplification products with a 1.8% agarose gel. Perform electrophoresis at 70 V for 30 min. Stain the gel in ethidium bromide solution for 10 min. View the gel under the Gel Doc EZ Imager (Bio-Rad, USA). Required oligos The protocol for the transformation by electroporation technique of C. vulgaris requires two PCR/RT-PCR primers. This set of primers targets the intergenic region between CamV 35s and the GUS gene (~ 120 bp) of the pCambia1303 vector to validate the successful transformation by electroporation technique, as shown in Table 1 . Table 1 PCR primers targeting the intergenic region between CamV 35s and GUS gene (~ 120 bp) of the pCambia1303 vector Primer Name Primer Sequences 5’–3’ Size (bp) P_Forward CTA TCC TTC GCA AGA CCC TTC C ~ 120 P_Reverse CAC GGG TTG GGG TTT CTA CAG Comments and troubleshooting It is important to ensure that high-quality RNA is obtained, as degraded RNA can lead to inefficient cDNA synthesis. In this study, the SuperScript™ IV First-Strand Synthesis System was used following the manufacturer’s instructions. To rule out gDNA contamination, include a no-reverse transcriptase control. For the PCR assay, prepare the reaction mix accurately, ensuring the concentrations of each component, i.e., Taq DNA polymerase, dNTPs, MgCl2, DNA template, and primers, are correct. If no amplification is observed, verify the sequence of the primers and check that the gDNA and cDNA are of good quality. Optimize the annealing temperature of the PCR conditions if necessary. The PCR conditions should be set precisely as indicated for amplifying the target region of the pCambia1303 (~ 120 bp). If non-specific bands are observed, consider increasing the annealing temperature and/or decreasing the working concentration of MgCl 2 . Resolving small-sized PCR products, i.e., ~ 120 bp, requires the use of a higher percentage of agarose gel. In this case, perform gel electrophoresis at 70 V for 30 mins at a 1.8% agarose gel. Following the completion of gel electrophoresis, stain the gel appropriately, ensuring safety precautions are followed if staining is done with ethidium bromide. The observation of PCR amplification products at the expected size suggests that a successful transformation was achieved. Nevertheless, we recommend validating the positive PCR amplicons through DNA sequencing. This is to confirm the identity of the amplicons is truly that of the transgene sequence of the pCambia1303 vector. Results The successful electroporation-mediated transformation of the C . vulgaris cell was first observed in the selective BG11 medium supplemented with 50 µg/mL of hygromycin B, as observed in Fig. 2 A. A negative control was included by using the wild-type C . vulgaris cultivated in the selective medium. Additionally, the wild-type C . vulgaris was also cultivated in a regular BG11 medium to ensure the feasibility of the medium being used. To further evaluate the successful transformation, molecular techniques, including gDNA isolation, RNA isolation, followed by PCR and RT-PCR assays targeting the integrated t-DNA region (intergenic region between CamV 35s and GUS gene), were employed. Figures 2 B and 2 C show the successful isolation of the gDNA and total RNA as observed in 1% agarose gel electrophoresis, while Fig. 2 D shows the positive amplifications of the PCR and RT-PCR assays at the expected size (~ 120 bp). Discussion The development of an effective transformation method on C. vulgaris is essential for genetic engineering in biotechnological research. The electroporation procedure presents a promising method of transformation of C. vulgaris . However, the rigidity of the C. vulgaris cell wall, which often requires pre-treatment prior to the transformation procedure, has caused inefficiency in the transformation process, leading to inherently low transformation rates. A critical factor in optimizing the electroporation parameter is the usage of a higher electric field strength (voltage). This will overcome the rigidity of the cell wall, increasing cell permeability and at the same time enhancing DNA uptake. The electroporation outlined here offers an efficient electroporation-mediated transformation without enzymatic treatment, providing a straightforward protocol to follow. This simplified procedure expands opportunities for research in microalgae genetic engineering, particularly for C. vulgaris . Conclusion In summary, an efficient and simple electroporation-mediated transformation method was developed for C . vulgaris . Declarations Conflict of Interest: The authors declare no competing interests. Author Contributions: All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by DMR and VSK. The first draft of the manuscript was written by DMR and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Ethical approval: All the procedures were carried out in accordance with the approval of the Department of Biosafety, Ministry of Natural Sources, Environment and Climate Change. JBK (S) 600-3/1/99. Funding: The work in this paper was supported by Universiti Malaysia Sabah, under the research grant DKC2010. References S. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7100769","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Method Article","associatedPublications":[],"authors":[{"id":485277642,"identity":"413a062c-c754-4836-91e7-62d8535837ed","order_by":0,"name":"Dexter Miller Robben","email":"","orcid":"","institution":"Universiti Malaysia Sabah, Jln UMS","correspondingAuthor":false,"prefix":"","firstName":"Dexter","middleName":"Miller","lastName":"Robben","suffix":""},{"id":485277643,"identity":"57ab5d7d-8ae0-4416-988c-8da91a8134e4","order_by":1,"name":"Zarina Amin","email":"","orcid":"","institution":"Universiti Malaysia Sabah, Jln UMS","correspondingAuthor":false,"prefix":"","firstName":"Zarina","middleName":"","lastName":"Amin","suffix":""},{"id":485277644,"identity":"9e9ac017-1035-4593-ad20-a27fed32a406","order_by":2,"name":"Cahyo Budiman","email":"","orcid":"","institution":"Universiti Malaysia Sabah, Jln UMS","correspondingAuthor":false,"prefix":"","firstName":"Cahyo","middleName":"","lastName":"Budiman","suffix":""},{"id":485277645,"identity":"c8c9b1ff-9c3c-46cf-8a92-edf3f0fc6bbd","order_by":3,"name":"Vijay Subbiah Kumar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvklEQVRIiWNgGAWjYDACZoYEBoYKCFuCBC1nSNICAoxtpGgxOM7w8DPvvMPR8g3MB2/zMGxLbCCo5TBDsjTvtsO5Gw6wJVvzMNwmrEWymSEBooWBx0yaWC3Jv3nnHM6d38D/jTgt/MwMadK8DYdzGw7wsBGvxXLOsfTcDYfZjC3nGNw2JqiFjf9M8o03Nda589ubH954U3FblqAWBgaeBCYeEM0MIgwYHInQwn6A8QcS156wjlEwCkbBKBhpAABKajrKhHAoHgAAAABJRU5ErkJggg==","orcid":"","institution":"Universiti Malaysia Sabah, Jln UMS","correspondingAuthor":true,"prefix":"","firstName":"Vijay","middleName":"Subbiah","lastName":"Kumar","suffix":""}],"badges":[],"createdAt":"2025-07-11 10:38:32","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7100769/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7100769/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11033-025-11167-x","type":"published","date":"2025-10-29T15:57:26+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":86859145,"identity":"4069bf54-9beb-43e7-82c6-aeb653213717","added_by":"auto","created_at":"2025-07-16 11:52:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":593600,"visible":true,"origin":"","legend":"\u003cp\u003eOverview workflow of the optimized transformation by electroporation technique of \u003cem\u003eC. vulgaris\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7100769/v1/76da38a745dc47ca3706c989.png"},{"id":86859136,"identity":"c48c4534-85fa-4f75-bc30-2b52fdf2a26b","added_by":"auto","created_at":"2025-07-16 11:52:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":96870,"visible":true,"origin":"","legend":"\u003cp\u003eSuccessful electroporation-mediated transformation of \u003cem\u003eC. vulgaris\u003c/em\u003e. A. Following the transformation procedure by electroporation technique and cultivation in regular BG11 medium during the resting period, transformed cells were cultivated in selective BG11 medium supplemented with 50 µg/mL of hygromycin B. Positive growth was observed in the transformed \u003cem\u003eC. vulgaris\u003c/em\u003e culture in the selective medium. 1 - wild type \u003cem\u003eC. vulgaris\u003c/em\u003ein regular BG11 medium; 2 - wild type \u003cem\u003eC. vulgaris\u003c/em\u003e in selective BG11 medium; 3 – transformed \u003cem\u003eC. vulgaris\u003c/em\u003e in selective BG11 medium. B. The transformed \u003cem\u003eC. vulgaris\u003c/em\u003e cells underwent genomic DNA isolation. Agarose gel electrophoresis (1%) showed successful isolation of the gDNA. C. The transformed \u003cem\u003eC. vulgaris\u003c/em\u003e cells underwent total RNA isolation. Agarose gel electrophoresis (1%) showed successful isolation of the total RNA. D. The isolated gDNA and RNA were subjected to PCR and RT-PCR assays, respectively. These assays targeted the intergenic region between the CamV 35s and GUS gene of the pCambia1303 vector with an approximate size of 120 bp. Successful amplification products were observed at the expected size in the agarose gel electrophoresis (1.8%).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7100769/v1/45812131f278b0bfb9bf75c6.png"},{"id":95040834,"identity":"cedb2695-26df-4e1c-bd16-27ac7bc7a85a","added_by":"auto","created_at":"2025-11-03 16:10:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1120243,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7100769/v1/15ab1077-8f3b-4206-879e-617ed1846d5b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eA Step-by-Step Approach to Establishing an Efficient Genetic Transformation Protocol for \u003cem\u003eChlorella vulgaris\u003c/em\u003e Using Electroporation\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMicroalgae are known for their diverse biotechnological applications, ranging from biofuel production to pharmaceuticals [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Additionally, the emergence of microalgae genetic engineering has provided a promising avenue for an alternative recombinant protein production system that is favourable over the traditional bacterial and yeast expression systems [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. \u003cem\u003eC. vulgaris\u003c/em\u003e, a green microalga belonging to the Chlorophyta family, has shown potential as a host organism for genetic transformation. The reason for this is due to its single-celled structure, which allows for a simple genetic transformation technique to be carried out. Additionally, the minimal cultivation requirements and rapid growth rate have allowed for the development of an alternative expression system with high scalability, while at the same time aligning with sustainable biotechnological practices.\u003c/p\u003e\u003cp\u003eOne of the most widely used transformation methods is electroporation. It facilitates the insertion of foreign genetic material into cells across diverse taxa, ranging from prokaryotes to eukaryotes. This method of transformation utilizes electrical pulses to create pores through the membrane of the cell, allowing an opening for foreign DNA to enter the cell. Consequently, the foreign DNA is incorporated into the genome of the host cell [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn \u003cem\u003eChlorella sp.\u003c/em\u003e, it is essential to establish an efficient genetic transformation method to further advance its applications in fundamental research in algal biotechnology. These transgenic lines of \u003cem\u003eC. vulgaris\u003c/em\u003e may play a significant role in the current biotechnological applications through their enhanced and improved yield and nutritional value, or their capability as a biological factory to produce a desired protein [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Notwithstanding, the recalcitrant nature of the \u003cem\u003eC. vulgaris\u003c/em\u003e cell wall poses a major challenge to a successful transformation. Due to its thick cell wall (15\u0026ndash;20 nm), \u003cem\u003eC. vulgaris\u003c/em\u003e is often treated with a mixture of enzymes to generate protoplasts prior to the transformation procedure [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. This limitation has hampered progress in genetic engineering efforts for \u003cem\u003eC. vulgaris.\u003c/em\u003e As a result, the development of an efficient transformation method remains vital for advancing research involving this species.\u003c/p\u003e\u003cp\u003eThis methodology paper details the procedure for efficient electroporation-mediated transformation of \u003cem\u003eC. vulgaris.\u003c/em\u003e Optimized electroporation parameters, recovery conditions, and the selection of transformants were highlighted. The described electroporation procedure provides a streamlined and effective approach for generating a transgenic line of \u003cem\u003eC. vulgaris\u003c/em\u003e strains, providing a robust tool for researchers to further study the biotechnological potential of this green microalga.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cem\u003eC. vulgaris\u003c/em\u003e culture was obtained from Algae Research Supply (Carlsbad, California), and all procedures related to the transformation of recombinant DNA and the cultivation of the transgenic \u003cem\u003eC. vulgaris\u003c/em\u003e were performed in a containment facility (Transgenic facility of Biotechnology Research Institute, Universiti Malaysia Sabah). Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows an overview of the entire electroporation-mediated transformation workflow.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eStep-by-step protocol\u003c/b\u003e\u003c/p\u003e\u003cp\u003eStep 1: Cultivation of \u003cem\u003eC. vulgaris\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eCultivate \u003cem\u003eC. vulgaris\u003c/em\u003e in BG11 medium (pH 6.8) at an ambient temperature of 28\u0026deg;C under a 16:8 photoperiod in a growth chamber.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eSubculture 10 mL of \u003cem\u003eC. vulgaris\u003c/em\u003e mother culture to 40 mL of BG11 medium under the same conditions as in the previous step.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eUse \u003cem\u003eC. vulgaris\u003c/em\u003e cells in the early growth phase of 7 days after subculture for the transformation by electroporation procedure.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003eComments and troubleshooting\u003c/p\u003e\u003cp\u003eIn this study, the BG11 medium was used for the cultivation of \u003cem\u003eC. vulgaris\u003c/em\u003e. It is important to monitor the pH of the BG11 medium regularly, as deviations from the optimal range of approximately pH 6.8 can impact the culture growth. In addition, temperature should be held consistently at 28\u0026deg;C, and the light regime follows a 16:8 photoperiod. We recommend manually agitate the culture once a day by gently swirling if a shaker is not available, as this will facilitate the dispersed cells to be uniformly exposed to light and nutrients.\u003c/p\u003e\u003cp\u003eWhen preparing the culture for electroporation, it is important to subculture the cells from the mother culture into fresh BG11 medium and allow them to grow for 7 days. This 7-day-old subculture, rather than the mother culture itself, should be used for the electroporation procedure. This is because cells in the early growth phase are more likely to have thinner cell walls and higher metabolic activity, making them more amenable to transformation. Overgrown cultures that have entered the stationary phase often have thicker cell walls, which can reduce transformation efficiency [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTo prevent contamination, it is crucial to practice aseptic techniques. If contamination arises, the affected culture should be discarded, and a new culture should be initiated.\u003c/p\u003e\u003cp\u003eStep 2: Preparation of \u003cem\u003eC. vulgaris\u003c/em\u003e cells\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eHarvest cells (1\u0026times;10\u003csup\u003e7\u003c/sup\u003e \u0026minus;\u0026thinsp;1\u0026times;10\u003csup\u003e8\u003c/sup\u003e cells per mL) through centrifugation at 10,000 rpm for 2 min at 4\u0026deg;C.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eWash the pelleted cells with 1 mL of 384 mM D-sorbitol at 2,500 rpm for 5 min at 4\u0026deg;C.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eRepeat the washing step three more times.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003eComments and troubleshooting\u003c/p\u003e\u003cp\u003eWhen preparing \u003cem\u003eC. vulgaris\u003c/em\u003e cells, ensure that the cell density is within the range of 1\u0026times;10⁷ \u0026ndash; 1\u0026times;10⁸ cells per mL before harvesting [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Too few cells may result in low transformation efficiency, while too many cells can lead to clumping and uneven washing. Follow the specified speed and conditions of the centrifugation to avoid cell damage. The higher speed (10,000 rpm) during harvesting ensures efficient pelleting, while the lower speed (2,500 rpm) during washing helps maintain cell integrity.\u003c/p\u003e\u003cp\u003eWashing steps with 384 mM D-sorbitol are critical to remove residual salts, which can interfere with electroporation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In this study, we recommend that the washing of the cells be done 4 times to ensure a thorough washing of the cells for an efficient transformation by electroporation. Maintaining sterile conditions throughout the procedure is also essential to avoid contamination. Use sterile tubes, pipettes, and solutions, and work in a clean environment to ensure.\u003c/p\u003e\u003cp\u003eStep 3: Transformation by electroporation technique\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eAdd 1 mL of 384 mM D-sorbitol to the washed cells and resuspend by pipetting up and down.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eAdd 1 \u0026micro;g of pCambia1303 vector and mix by gently flicking the tube.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eTransfer the mixture to an electroporation cuvette and incubate the mixture on ice for 10 min.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003ePlace the cuvette in the electroporation pod and subject it to a single-pulse electroporation with an electric field of 2.2 kV, a capacitance of 50 \u0026micro;F, and a resistance of 500 Ω using a Bio-Rad Gene Pulser II (Biorad, USA).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003ePlace the cuvette and incubate the mixture on ice for 10 min.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eTransfer electroporated cells to a 15 mL Falcon tube containing 10 mL of BG11 medium and incubate the cells at room temperature for two days in low light exposure.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003eComments and troubleshooting\u003c/p\u003e\u003cp\u003eIt is crucial to ensure that the cells to be transformed are resuspended evenly in 384 mM D-sorbitol. Resuspension should be done by gently pipetting up and down until a homogenous mixture is observed. The use of a high quantity of plasmid DNA is also important to ensure a high possibility of the DNA being taken up by the \u003cem\u003eC. vulgaris\u003c/em\u003e cells. In this study, we use the pCambia1303 vector at a concentration of 1 \u0026micro;g. Cooling of cells before and after electroporation is essential to maintain the permeability of the cell membrane and improve the DNA uptake. Skipping this step or insufficient cooling can lead to poor transformation results. We recommend an incubation period of the \u003cem\u003eC. vulgaris\u003c/em\u003e cells (before and after electroporation) on ice for 10 min.\u003c/p\u003e\u003cp\u003eIn this study, the electroporation parameters used are 2.2 kV, 50 \u0026micro;F, 500 Ω. These parameters are effective for the transformation of \u003cem\u003eC. vulgaris\u003c/em\u003e without prior enzyme treatment. Due to the thick cell wall of \u003cem\u003eC. vulgaris\u003c/em\u003e, a high voltage is employed. Arcing is a common occurrence in electroporation procedures that is usually caused by the presence of salts. Thus, it is important to ensure that the cells are sufficiently washed to remove residual salts. If transformation efficiency is low, consider optimizing the parameters for your specific setup. Following the electroporation of the \u003cem\u003eC. vulgaris\u003c/em\u003e cells and an incubation period on ice, immediately transfer the cells to BG11 medium to minimize stress and allow for cell recovery [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. This step is known as the resting period post-transformation by electroporation. It is recommended that the incubation period be two days at room temperature under low light. If cell viability is low after electroporation, try reducing the electric field strength slightly or increasing the recovery time.\u003c/p\u003e\u003cp\u003eStep 4: Cultivation of transformed \u003cem\u003eC. vulgaris\u003c/em\u003e in selective medium\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eFollowing the 2-day resting period, subculture 100 \u0026micro;L of the transformed \u003cem\u003eC. vulgaris\u003c/em\u003e culture to a 5 mL BG11 medium supplemented with 50 \u0026micro;g/mL of hygromycin B.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eMaintain the culture conditions at an ambient temperature of 28\u0026deg;C under a 16:8 photoperiod in a growth chamber.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eObserve for growth daily. Growth is expected to be visibly observed after seven days of cultivation.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003eComments and troubleshooting\u003c/p\u003e\u003cp\u003eIt is important to use the correct concentration of hygromycin B (50 \u0026micro;g/mL) when cultivating the transformed \u003cem\u003eC. vulgaris\u003c/em\u003e. This is to ensure effective selection of the transformed cells. Avoid fluctuations of the culture conditions, such as temperature and light exposure, as these may cause stress to the cell and delay growth. If no growth is observed after 7 days, ensure that the antibiotic used has not expired and verify its concentration. Discard the culture if contamination is observed and restart the culture with sterile techniques.\u003c/p\u003e\u003cp\u003eLastly, increase the initial inoculum volume slightly or extend the growth period if growth is slower than expected. Some transformed cells may take longer to adapt to the selective medium. A lower concentration of antibiotic (i.e., 30 \u0026micro;g/mL) can be used at the initial subculture before using a higher concentration of antibiotic at the subsequent culture. Regular monitoring and adjustments will help ensure the successful cultivation of transformed \u003cem\u003eC. vulgaris\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eStep 5: Genomic DNA and total RNA isolation from the transformed \u003cem\u003eC. vulgaris\u003c/em\u003e cells\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eHarvest the transformed \u003cem\u003eC. vulgaris\u003c/em\u003e cells by centrifugation at rpm for min at 4\u0026deg;C.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eExtract the gDNA and total RNA of the harvested cells using the Wizard\u0026reg; Genomic DNA Purification Kit (Promega, USA) and PureLinkTM RNA Mini Kit (Thermo Fisher Scientific, USA) following the manufacturer\u0026rsquo;s protocol, respectively.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eElectrophorese the isolated gDNA and total RNA in 1% agarose gel. Perform electrophoresis at 70 V for 30 min.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eStain the gel in ethidium bromide solution for 10 min. View the gel under the Gel Doc EZ Imager (Bio-Rad, USA).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003eComments and troubleshooting\u003c/p\u003e\u003cp\u003eThe use of other commercially available kits for the gDNA and RNA extractions is feasible. However, it is important to ensure the compatibility of the kit to the type of sample under study. In this study, the Wizard\u0026reg; Genomic DNA Purification Kit (Promega, USA) and PureLinkTM RNA Mini Kit (Thermo Fisher Scientific, USA) were used for the isolation of the gDNA and RNA from wild-type and transformed \u003cem\u003eC. vulgaris\u003c/em\u003e cells.\u003c/p\u003e\u003cp\u003eWhen harvesting the \u003cem\u003eC. vulgaris\u003c/em\u003e cells, ensure cells are thoroughly centrifuged at the specified speed and time (10,000 rpm for 10 min at 4\u0026deg;C). The starting volume of the culture may be increased if the pellet obtained after centrifugation is too small. Follow the manufacturer\u0026rsquo;s instructions for the kits used for the isolation of nucleic acids to ensure a good-quality yield. Especially when isolating RNA, degradation can occur if proper precautions are not taken, such as using RNase-free consumables and working in an RNase-free environment.\u003c/p\u003e\u003cp\u003eFor resolving gDNA and RNA samples, we recommend using a 1% agarose gel to separate the nucleic acids effectively and perform the gel electrophoresis at 70 V for 30 min. Following the completion of the gel electrophoresis, stain the gel appropriately, ensuring safety precautions are followed if staining is done with ethidium bromide. Ensure that the intact bands of DNA and RNA are present before proceeding to the next step.\u003c/p\u003e\u003cp\u003eStep 6: PCR and RT-PCR assay targeting the pCambia1303 vector intergenic region (~\u0026thinsp;120 bp)\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eFor the RNA sample, perform cDNA synthesis using the SuperScript\u0026trade; IV First-Strand Synthesis System (Thermo Fisher Scientific, USA) following the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003ePrepare a 20 \u0026micro;l PCR mix with the following reagents to generate amplified copies of the gDNA and cDNAs: 1 \u0026micro;l of gDNA or cDNAs, 1 \u0026micro;l of Taq polymerase (5 U/\u0026micro;l, Promega, USA), 4 \u0026micro;l of 5\u0026times; PCR buffer, 0.4 \u0026micro;l dNTPs (10 mM), 1.2 \u0026micro;l MgCl2 (1.5 mM), 0.6 \u0026micro;l of each PCR primer (25 \u0026micro;M), and top up the volume with nuclease-free water.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eThe amplification program is set for an initial denaturation at 95\u0026deg;C for 5 min, with 35 cycles of denaturation at 95\u0026deg;C for 45 s, annealing at 55\u0026deg;C for 30 s, and extension at 72\u0026deg;C for 45 s, followed by final extension at 72\u0026deg;C for 5 min.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eAnalyse the PCR amplification products with a 1.8% agarose gel. Perform electrophoresis at 70 V for 30 min.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eStain the gel in ethidium bromide solution for 10 min. View the gel under the Gel Doc EZ Imager (Bio-Rad, USA).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eRequired oligos\u003c/span\u003e\u003c/p\u003e\u003cp\u003eThe protocol for the transformation by electroporation technique of \u003cem\u003eC. vulgaris\u003c/em\u003e requires two PCR/RT-PCR primers. This set of primers targets the intergenic region between CamV 35s and the GUS gene (~\u0026thinsp;120 bp) of the pCambia1303 vector to validate the successful transformation by electroporation technique, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePCR primers targeting the intergenic region between CamV 35s and GUS gene (~\u0026thinsp;120 bp) of the pCambia1303 vector\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePrimer Name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePrimer Sequences\u003c/p\u003e\u003cp\u003e5\u0026rsquo;\u0026ndash;3\u0026rsquo;\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSize (bp)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP_Forward\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCTA TCC TTC GCA AGA CCC TTC C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e~\u0026thinsp;120\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP_Reverse\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCAC GGG TTG GGG TTT CTA CAG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eComments and troubleshooting\u003c/p\u003e\u003cp\u003eIt is important to ensure that high-quality RNA is obtained, as degraded RNA can lead to inefficient cDNA synthesis. In this study, the SuperScript\u0026trade; IV First-Strand Synthesis System was used following the manufacturer\u0026rsquo;s instructions. To rule out gDNA contamination, include a no-reverse transcriptase control. For the PCR assay, prepare the reaction mix accurately, ensuring the concentrations of each component, i.e., Taq DNA polymerase, dNTPs, MgCl2, DNA template, and primers, are correct. If no amplification is observed, verify the sequence of the primers and check that the gDNA and cDNA are of good quality. Optimize the annealing temperature of the PCR conditions if necessary.\u003c/p\u003e\u003cp\u003eThe PCR conditions should be set precisely as indicated for amplifying the target region of the pCambia1303 (~\u0026thinsp;120 bp). If non-specific bands are observed, consider increasing the annealing temperature and/or decreasing the working concentration of MgCl\u003csup\u003e2\u003c/sup\u003e. Resolving small-sized PCR products, i.e., ~\u0026thinsp;120 bp, requires the use of a higher percentage of agarose gel. In this case, perform gel electrophoresis at 70 V for 30 mins at a 1.8% agarose gel. Following the completion of gel electrophoresis, stain the gel appropriately, ensuring safety precautions are followed if staining is done with ethidium bromide. The observation of PCR amplification products at the expected size suggests that a successful transformation was achieved. Nevertheless, we recommend validating the positive PCR amplicons through DNA sequencing. This is to confirm the identity of the amplicons is truly that of the transgene sequence of the pCambia1303 vector.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe successful electroporation-mediated transformation of the \u003cem\u003eC\u003c/em\u003e. \u003cem\u003evulgaris\u003c/em\u003e cell was first observed in the selective BG11 medium supplemented with 50 \u0026micro;g/mL of hygromycin B, as observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA. A negative control was included by using the wild-type \u003cem\u003eC\u003c/em\u003e. \u003cem\u003evulgaris\u003c/em\u003e cultivated in the selective medium. Additionally, the wild-type \u003cem\u003eC\u003c/em\u003e. \u003cem\u003evulgaris\u003c/em\u003e was also cultivated in a regular BG11 medium to ensure the feasibility of the medium being used. To further evaluate the successful transformation, molecular techniques, including gDNA isolation, RNA isolation, followed by PCR and RT-PCR assays targeting the integrated t-DNA region (intergenic region between CamV 35s and GUS gene), were employed. Figures\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC show the successful isolation of the gDNA and total RNA as observed in 1% agarose gel electrophoresis, while Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD shows the positive amplifications of the PCR and RT-PCR assays at the expected size (~\u0026thinsp;120 bp).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe development of an effective transformation method on \u003cem\u003eC. vulgaris\u003c/em\u003e is essential for genetic engineering in biotechnological research. The electroporation procedure presents a promising method of transformation of \u003cem\u003eC. vulgaris\u003c/em\u003e. However, the rigidity of the \u003cem\u003eC. vulgaris\u003c/em\u003e cell wall, which often requires pre-treatment prior to the transformation procedure, has caused inefficiency in the transformation process, leading to inherently low transformation rates. A critical factor in optimizing the electroporation parameter is the usage of a higher electric field strength (voltage). This will overcome the rigidity of the cell wall, increasing cell permeability and at the same time enhancing DNA uptake. The electroporation outlined here offers an efficient electroporation-mediated transformation without enzymatic treatment, providing a straightforward protocol to follow. This simplified procedure expands opportunities for research in microalgae genetic engineering, particularly for \u003cem\u003eC. vulgaris\u003c/em\u003e.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, an efficient and simple electroporation-mediated transformation method was developed for \u003cem\u003eC\u003c/em\u003e. \u003cem\u003evulgaris\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by DMR and VSK. The first draft of the manuscript was written by DMR and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval:\u003c/strong\u003e All the procedures were carried out in accordance with the approval of the Department of Biosafety, Ministry of Natural Sources, Environment and Climate Change. JBK (S) 600-3/1/99.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e The work in this paper was supported by Universiti Malaysia Sabah, under the research grant DKC2010.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eS. Sigamani, D. Ramamurthy, and H. Natarajan, \u0026ldquo;A Review on Potential Biotechnological applications of Microalgae,\u0026rdquo; \u003cem\u003eJ Appl Pharm Sci\u003c/em\u003e, vol. 6, no. 8, pp. 179\u0026ndash;184, Aug. 2016, doi: 10.7324/JAPS.2016.60829.\u003c/li\u003e\n\u003cli\u003eM. G. Morais, T. D. Santos, L. Moraes, B. S. Vaz, E. G. Morais, and J. A. V. Costa, \u0026ldquo;Exopolysaccharides from microalgae: Production in a biorefinery framework and potential applications,\u0026rdquo; \u003cem\u003eBioresour Technol Rep\u003c/em\u003e, vol. 18, p. 101006, Jun. 2022, doi: 10.1016/J.BITEB.2022.101006.\u003c/li\u003e\n\u003cli\u003eK. Mulluye, Y. Bogale, D. Bayle, and Y. Atnafu, \u0026ldquo;Review on Microalgae Potential Innovative Biotechnological Applications,\u0026rdquo; \u003cem\u003eBiosci Biotechnol Res Asia\u003c/em\u003e, vol. 20, no. 1, pp. 35\u0026ndash;43, Mar. 2023, doi: 10.13005/BBRA/3066.\u003c/li\u003e\n\u003cli\u003eM. I. Khan, J. H. Shin, and J. D. Kim, \u0026ldquo;The promising future of microalgae: Current status, challenges, and optimization of a sustainable and renewable industry for biofuels, feed, and other products,\u0026rdquo; Mar. 05, 2018, \u003cem\u003eBioMed Central Ltd.\u003c/em\u003e doi: 10.1186/s12934-018-0879-x.\u003c/li\u003e\n\u003cli\u003eS. B. Grama, Z. Liu, and J. Li, \u0026ldquo;Emerging Trends in Genetic Engineering of Microalgae for Commercial Applications,\u0026rdquo; \u003cem\u003eMar Drugs\u003c/em\u003e, vol. 20, no. 5, May 2022, doi: 10.3390/MD20050285,.\u003c/li\u003e\n\u003cli\u003eL. Mu\u0026ntilde;oz-Sol\u0026oacute;rzano, K. Willis-Ure\u0026ntilde;a, S. Valverde-Rojas, M. Jarqu\u0026iacute;n-Cordero, and L. Barboza-Fallas, \u0026ldquo;Microalgae as expression systems for recombinant protein production,\u0026rdquo; \u003cem\u003eRevista Tecnolog\u0026iacute;a en Marcha\u003c/em\u003e, Nov. 2024, doi: 10.18845/TM.V37I9.7608.\u003c/li\u003e\n\u003cli\u003eM. Kumar, J. Jeon, J. Choi, and S. R. Kim, \u0026ldquo;Rapid and efficient genetic transformation of the green microalga Chlorella vulgaris,\u0026rdquo; \u003cem\u003eJ Appl Phycol\u003c/em\u003e, vol. 30, no. 3, pp. 1735\u0026ndash;1745, Jun. 2018, doi: 10.1007/s10811-018-1396-3.\u003c/li\u003e\n\u003cli\u003eZ. X. Chong, S. K. Yeap, and W. Y. 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D. Tripp, J. L. Lilley, W. N. Wood, and L. K. Lewis, \u0026ldquo;Enhancement of plasmid DNA transformation efficiencies in early stationary-phase yeast cell cultures,\u0026rdquo; \u003cem\u003eYeast\u003c/em\u003e, vol. 30, no. 5, pp. 191\u0026ndash;200, May 2013, doi: 10.1002/YEA.2951.\u003c/li\u003e\n\u003cli\u003eO. Kilian, C. S. E. Benemann, K. K. Niyogi, and B. Vick, \u0026ldquo;High-efficiency homologous recombination in the oil-producing alga Nannochloropsis sp.,\u0026rdquo; \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e, vol. 108, no. 52, pp. 21265\u0026ndash;21269, Dec. 2011, doi: 10.1073/PNAS.1105861108/SUPPL_FILE/PNAS.201105861SI.PDF.\u003c/li\u003e\n\u003cli\u003eA. A. Zainal Abidin, M. Suntarajh, and Z. N. Balia Yusof, \u0026ldquo;Transformation of a Malaysian species of Nannochloropsis: gateway to construction of transgenic microalgae as vaccine delivery system to aquatic organisms,\u0026rdquo; \u003cem\u003eBioengineered\u003c/em\u003e, vol. 11, no. 1, pp. 1071\u0026ndash;1079, Jan. 2020, doi: 10.1080/21655979.2020.1822106.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"microalgae, genetic engineering, electroporation, recombinant DNA","lastPublishedDoi":"10.21203/rs.3.rs-7100769/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7100769/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eChlorella vulgaris\u003c/em\u003e, a unicellular green microalgae, has shown diverse applications in biotechnology. Particularly in microalgae genetic engineering, genetic transformation of \u003cem\u003eC. vulgaris\u003c/em\u003e offers a sustainable and cost-effective approach to producing recombinant protein. Despite this, the thick cell wall of \u003cem\u003eC. vulgaris\u003c/em\u003e poses a challenge, often necessitating optimization of the transformation parameters to overcome the challenge. This current study presents a detailed methodology for efficient electroporation-mediated transformation of \u003cem\u003eC. vulgaris\u003c/em\u003e. The electroporation procedure described here includes \u003cem\u003eC. vulgaris\u003c/em\u003e cell preparation and electroporation parameters such as pulse voltage, capacitance, and resistance. Prior to the cultivation of the transformed cells in a selective BG11 medium, recovery conditions of the newly transformed cells in a regular BG11 medium were also described. Through optimization of these parameters, an improved transformation efficiency while maintaining cell viability was achieved. Positive amplifications of the pCambia1303 t-DNA region in the PCR and RT-PCR assays were indicative of the success of the electroporation-mediated transformation of \u003cem\u003eC. vulgaris\u003c/em\u003e. DNA sequencing was then used to confirm the identity of these amplicons, thus validating the successful transformation procedure. The electroporation-mediated transformation procedure established here provides a reliable and reproducible method for carrying out genetic modification on \u003cem\u003eC. vulgaris\u003c/em\u003e to facilitate its potential use as a bio-factory in numerous biotechnological applications.\u003c/p\u003e","manuscriptTitle":"A Step-by-Step Approach to Establishing an Efficient Genetic Transformation Protocol for Chlorella vulgaris Using Electroporation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-16 11:52:27","doi":"10.21203/rs.3.rs-7100769/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-04T14:09:34+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-01T16:18:45+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-31T13:55:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-24T11:31:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"47035170921514044531511809312928301970","date":"2025-07-21T07:06:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"101720697808212915354826693538988876522","date":"2025-07-16T17:24:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"166411750649370601130135461183903124935","date":"2025-07-16T10:49:05+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-14T15:30:21+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-12T14:03:01+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-12T14:02:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Biology Reports","date":"2025-07-11T10:33:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a11c2b8b-5092-4f01-b6f9-beea6557f175","owner":[],"postedDate":"July 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-03T16:08:09+00:00","versionOfRecord":{"articleIdentity":"rs-7100769","link":"https://doi.org/10.1007/s11033-025-11167-x","journal":{"identity":"molecular-biology-reports","isVorOnly":false,"title":"Molecular Biology Reports"},"publishedOn":"2025-10-29 15:57:26","publishedOnDateReadable":"October 29th, 2025"},"versionCreatedAt":"2025-07-16 11:52:27","video":"","vorDoi":"10.1007/s11033-025-11167-x","vorDoiUrl":"https://doi.org/10.1007/s11033-025-11167-x","workflowStages":[]},"version":"v1","identity":"rs-7100769","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7100769","identity":"rs-7100769","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}