Genetically Modified Organisms Research Articles

Comparison of DNA isolation methods for GMO detection from biscuit samples

The global increase in genetically modified organism (GMO) content in feed and food products has necessitated the development of precise detection methods to differentiate between biotechnologically derived foods and those without GMOs. Despite the various regulations in different countries, an internationally consistent approach to labeling GMO products is needed. For this reason, there is a widespread need to develop effective GMO detection methods to provide reliable and transparent food safety to consumers. The first experimental step in creating accurate and reliable detection methods for GMOs is effective DNA isolation. Determining DNA isolation methods specific to different processing levels of foods is very important. This study was aimed to compare different DNA extraction methods in biscuit samples. For this reason, DNA from different biscuit samples was isolated using three different protocols (CTAB-PVP, modified CTAB-1, and modified CTAB-2). DNA samples extracted from biscuits, were evaluated regarding DNA concentration and purity. According to the results obtained, the CTAB-2 procedure generally showed the best performance in terms of both DNA amount and purity rates for biscuit samples.

Does DNA extraction affect the specificity of a PCR method claiming the specific detectability of a genome-edited plant?

ABSTRACT Under current EU legislation, genetically modified organisms (GMOs) and derived food and feed products must be authorized as GM food, feed, or seed and appropriate detection methods must be made available for use in official controls. A Real-Time PCR method has recently been published by Chhalliyil et al. claiming to be specific for the detection and identification of genome-edited oilseed rape (OSR) lines commercialized in North America. In a previous study, we have independently assessed this method in three reference laboratories for sensitivity, specificity, and robustness. We found that the method does not meet all the minimum performance requirements (MPR) for GMO testing in the EU, which contradicts the claims of the method developer. Here we show, in addition to the previously published method assessment study that a modified DNA extraction is not the reason for the contradictory findings and does not affect the specificity of the method. We also discuss the procedures recently proposed by the method developers for interpreting PCR results with high Cq values.

Enhancing Environmental Conservation through the Implementation of GMOs

The rapid advancements in genetic engineering have opened new frontiers in agriculture, medicine, and environmental conservation. Among these, the potential of genetically modified organisms (GMOs) in preserving biodiversity and combating environmental challenges has garnered significant attention. This article delves into how GMOs contribute to conservation efforts by bolstering species resilience and curbing biodiversity decline. For instance, the case of ash trees in North America facing the threat of emerald ash borers showcases how genetic modification can aid in rescuing endangered species. The discussion also delves into measures overseeing GMO releases into environments highlighting approaches taken by countries such as the U.S. And China to mitigate risks such as diminished genetic diversity and ecological disturbances. While GMOs offer advantages like lowering greenhouse gas emissions and aiding in toxin breakdown, there are concerns about drawbacks such as displacing species or causing unintended ecological consequences. To address these issues innovative solutions are proposed—like using GMOs that self-terminate after their intended purpose or creating GMOs that establish symbiotic relationships with species.

Latin America: A hub for agrobiotechnological innovations.

Modern biotechnology is one of the last century's major advances in human science. Particularly in the agronomical field, the landscape of crop improvement technologies has witnessed a great expansion, driven by the integration of molecular and genetic engineering methodologies into the breeding toolbox. Latin America (LATAM) serves as a pioneering region in incorporating such techniques with several countries swiftly embracing these technologies. This review aims to give a comprehensive overview of the elements that influenced agrobiotech acceptance in LATAM countries and how such cases could provide support for upcoming technologies to be considered worldwide. Nearly 50 years of biotech breakthroughs have provided humankind with an impressive portfolio of tools already integrated into several life-sciences areas. The agronomical field has greatly progressed thanks to technologies derived from Genetically Modified Organisms (GMOs) and high promises are being made to also incorporate genome -editing products. LATAM's case is a prime example of how early introduction of novelties in the crop production chain can result in improved yields, paving the way for future developments to be easily integrated into the technological ecosystem of a region. The example set by LATAM can also be useful for the present gene-editing regulatory scenario. With several countries presently on the path to approving these methods in their current crop systems, basing their next steps on the southern continent's example, could represent a safe and practical pathway towards a new agronomical revolution.

Microhomology-mediated end-joining Knock-In approaches to delete the allergenic domain of trout parvalbumin beta-1. Preliminary results in F0 animals and feedback

Background Gene editing techniques offer new opportunities to improve important traits in aquaculture. The allergenicity of fish flesh is a major problem in aquaculture. Parvalbumin (Parv) is the most prevalent fish allergen. For instance, in salmonids, a single parvalbumin beta-1 protein (parvb1) has been identified as an allergen in specific patients. Therefore, generating trout carrying two parvb1 alleles deleted from the allergenic peptide-encoding region could prevent allergies in these sensitive individuals. Methods Here, we describe the application of the Crispr/cas9 system in an attempt to delete parvb1 exon 2 encoding the allergenic peptide and, alternatively, to replace exon 2 of parvb1 with exon2 of parvalbumin beta-2 protein (parvb2,) which does not encode the allergenic peptide. Exon skipping and swapping were pursued through microhomology-mediated end-joining (MMEJ) knock-In using specifically designed double-stranded donor DNA. Results Genotyping of approximately 200 F0 fingerlings originating from eggs injected with donor DNA designed for exon 2 skipping led to the identification of only one animal carrying an allele lacking exon 2. Genotyping of approximately 150 fingerlings originating from eggs injected with donor DNA for exon 2 swapping did not result in any trout carrying the expected modified allele. Conclusions These preliminary results indicate the potential difficulties associated with the MMEJ KI experiments performed in farmed fish. Finally, new genomic techniques in aquaculture are further discussed in the context of lively debates taking place in the European parliament regarding a possible revision of the current law that determines the legal status of farm animals modified by genome editing. Gene editing, microhomology-mediated end-joining knock-in, parvalbumin, allergenicity, trout, and genetically modified organisms (GMOs).

Regulation of genome edited organismsin Australia.

Whether organisms developed with the use of genome editing techniques, or food derived from such organisms, are, or should be, regulated as genetically modified organisms (GMOs) or genetically modified (GM) food, respectively, remains a subject of debate globally. Much of the discussion has been scientific and focussed on the similar genetic outcomes of some genome editing techniques and 'conventional' or natural mutagenesis. Many jurisdictions, including Australia, have considered, or are considering, how their regulatory frameworks will deal with such organisms and products. In Australia, organisms developed with site directed nuclease 1 (SDN-1, with no added template to guide homology-directed repair) are not regulated as GMOs, pursuant to exclusions in the Gene Technology Regulations 2001. The exclusion of SDN-1 organismsfrom regulation in Australia is sometimes misrepresented, including in scientific peer reviewed publications, as extending to all genome edited organisms. This highlights the importance for researchers, developers and other stakeholders to understand that whether genome edited organisms are, or are not, subject to regulation as GMOs in a particular jurisdiction may quintessentially be a legal question, not a scientific one.

Organic Farming for Residue-Free Production

Organic farming has emerged as a sustainable agricultural practice aimed at producing food without synthetic chemicals, pesticides, and genetically modified organisms (GMOs). This approach promotes biodiversity, soil health, and environmental sustainability while ensuring residue-free food production. The key principles of organic farming include crop rotation, composting, natural pest management, and the use of organic inputs such as manure and biological controls. These practices not only enhance soil fertility and biodiversity but also contribute to mitigating climate change by sequestering carbon in soils. Residue-free production in organic farming is achieved through rigorous adherence to organic standards and certification processes. Organic certification ensures that farmers comply with strict regulations regarding inputs, practices, and traceability throughout the production chain. Consumers increasingly prefer organic products due to concerns about chemical residues in food and their potential impact on health. Organic farming promotes transparency and consumer trust by offering products that are free from synthetic pesticides, herbicides, and fertilizers. The global market for organic products continues to expand, driven by growing consumer awareness and demand for healthier and environmentally friendly food options. Organic farming presents opportunities for farmers to access premium markets, improve farm profitability, and contribute to sustainable agriculture. However, challenges such as certification costs, market access barriers, and competition with conventional agriculture remain significant. Future research and development in organic farming are crucial to addressing these challenges and enhancing the scalability and efficiency of residue-free production. Innovations in organic pest management, soil health management, and sustainable agriculture practices will further support the growth of organic farming worldwide. Overall, organic farming stands as a promising solution for residue-free food production, promoting human health, environmental stewardship, and sustainable development in agriculture.

Deep learning assisted, smartphone based universal Multi-RPA-CRISPR/Cas12a-G4 portable chip for simultaneous detection of CaMV35S and NOS

Portable and fast genetically modified organisms (GMOs) detection can achieve accurate labeling and precise regulation. Here, we build a universal and intelligent Multi-RPA-CRISPR/Cas12a-G4 detection chip platform for simultaneous CaMV35S and NOS detection. The use of multiple primer pairs for RPA enables one-tube amplification of dual targets. Independent cleavage of Cas12a for dual targets was controlled by flow amplification products into the respective detection chambers containing CaMV35S crRNA and NOS crRNA. The detection results are obtained based that uncut G4 in solution can produce peroxidase activity to catalyze ABTS2− making the negative result approaching dark green. The multi-RPA-CRISRP/Cas12a-G4 colorimetric platform achieves a 1 aM LOD for CaMV35S and NOS in 55 min. The Yolov5 deep learning algorithm and the analysis of gray values was trained and developed for colorimetric detection that is more sensitive than the naked eye. These algorithms are integrated into a smartphone app to automate the reading of test results. The 100% accuracy of colorimetry detection and the 93.3% accuracy of portable chip detection for rice and soy sauce samples demonstrate our method can rapidly and inexpensively detect samples of GMOs across species. Overall, our efficient, low-cost portable Multi-RPA-CRISPR/Cas12a-G4 detection platform coupled with deep learning algorithms enables portable GMOs detection with automated signal readout.

Screening for contamination with genetically modified organisms in food, feed and their additives obtained in retail and online shops in Latvia

The aim of the study was to screen for contamination of food and feed products with genetically modified organisms (GMO) in several sample groups obtained from retail and online shops. The total number of samples was 171, including 67 of food, 26 feed, 27 additive, and 51 samples of dried pollen for human consumption. Detection and quantification of GMO in food and feed was performed according to the approved quantitative real-time PCR methods. Fermentation product samples were analyzed using screening for antimicrobial resistance genes, 16S RNA and ITS1 sequencing, isolation of bacterial cultures and whole genome sequencing. In total, GMO contamination was found in 12 samples (in 10.45% of food and in 19.23% of feed samples). A lower proportion of GMO containing samples were from the European Union (10.26 %) vs. samples from outside (17.02%), but none with ingredients of both origins. Non-compliant were three food and five feed samples. Of the 27 samples of food and feed additives and food supplements, 10 samples were possibly non-compliant, as they contained live bacteria with antimicrobial resistance genes, and seven of them contained possibly recombinant DNA. The study indicates presence of GMO contamination that emphasizes the need for further monitoring.

Review of the technology used for structural characterization of the GMO genome using NGS data

The molecular characterization of genetically modified organisms (GMOs) is essential for ensuring safety and gaining regulatory approval for commercialization. According to CODEX standards, this characterization involves evaluating the presence of introduced genes, insertion sites, copy number, and nucleotide sequence structure. Advances in technology have led to the increased use of next-generation sequencing (NGS) over traditional methods such as Southern blotting. While both methods provide high reproducibility and accuracy, Southern blotting is labor-intensive and time-consuming due to the need for repetitive probe design and analyses for each target, resulting in low throughput. Conversely, NGS facilitates rapid and comprehensive analysis by mapping whole-genome sequencing (WGS) data to plasmid sequences, accurately identifying T-DNA insertion sites and flanking regions. This advantage allows for efficient detection of T-DNA presence, copy number, and unintended gene insertions without additional probe work. This paper reviews the current status of GMO genome characterization using NGS and proposes more efficient strategies for this purpose.

Advances in Genotyping Detection of Fragmented Nucleic Acids.

Single nucleotide variant (SNV) detection is pivotal in various fields, including disease diagnosis, viral screening, genetically modified organism (GMO) identification, and genotyping. However, detecting SNVs presents significant challenges due to the fragmentation of nucleic acids caused by cellular apoptosis, molecular shearing, and physical degradation processes such as heating. Fragmented nucleic acids often exhibit variable lengths and inconsistent breakpoints, complicating the accurate detection of SNVs. This article delves into the underlying causes of nucleic acid fragmentation and synthesizes the strengths and limitations of next-generation sequencing technology, high-resolution melting curves, molecular probes, and CRISPR-based approaches for SNV detection in fragmented nucleic acids. By providing a detailed comparative analysis, it seeks to offer valuable insights for researchers working to overcome the challenges of SNV detection in fragmented samples, ultimately advancing the accurate and efficient detection of single nucleotide variants across diverse applications.

The Role of Heuristics and Biases in the Choice of Risk Triggers for Novel Foods and GMOs in the European Union

Abstract In the European Union (EU), novel foods and Genetically Modified Organisms (GMOs) are subject to long and costly authorisation procedures and post-marketing requirements. The regulatory frameworks applicable to novel foods and GMOs come into effect based on perceived factors of risk. These “risk triggers” are characteristics of novel foods and GMOs which differentiate them from traditional foods, creating a presumption of risk. Within the EU, consumer acceptance of genetically modified foods and novel foods like insects or cultivated meat is shaped by heuristics and biases, mainly focusing on the “novelty” and “unnaturalness” of these products, resulting in a predominantly negative perception. This paper investigates the close connection between cognitive biases identified in consumer perception literature and the implementation of specific risk triggers in the regulation of novel foods and GMOs in the EU. It subsequently raises concerns about the appropriateness of these risk triggers in forming a presumption of risk for these innovative products.

Livestock farmers’ perceptions towards genetically engineered methods and genetically modified organisms in Denmark and Sweden

Although studies paid extensive attention to consumers' perceptions towards risk related to genetic engineering (GE) methods and genetically modified organisms (GMOs) in food, farmers' views have been under-researched. Understanding what shapes farmers’ perceptions towards potential risks by GE methods in food production matters, because as producers and suppliers of such food products, farmers affect their use and commercialisation. Scientists have debated and disagreed on the safety of GE methods and GMOs and their impact on biodiversity and environment over the years. These disagreements impeded the extensive GMO food production in the EU, which is also reflected in consumers' perceptions about potential risks by using GE methods concerning health safety and nutritional value of GMO food.1.This paper does not evaluate the potential benefits or hazards of these methods. Instead, based on primary survey data, it investigates and compares the aspects livestock farmers perceive as important towards GE methods and GMOs in Denmark and Sweden. The analysis demonstrates that farmers’ perceptions towards potential risks using GE methods and GMOs differ among types of producers within each country and between the two countries. These differences in perceptions are attributed to socio-economic characteristics (age, education, economic benefits, and farming activities), sectoral organisation, trust in suppliers and the social environment within which farmers operate in rural areas.

Gene Editing for Disease Resistance in Crops: Success Stories and Challenges

Gene editing has emerged as a transformative tool in modern agriculture, offering new avenues for enhancing disease resistance in crops. By precisely modifying the DNA of plants, scientists can develop varieties that are better equipped to withstand the onslaught of pathogens, which pose significant threats to global food security. This article delves into the success stories and challenges associated with gene editing for disease resistance in crops, with a focus on technologies like CRISPR-Cas9, TALENs, and ZFNs. One of the major success stories in this field is the development of disease-resistant varieties of wheat, rice, and tomatoes. For instance, researchers have used CRISPR-Cas9 to create wheat varieties resistant to powdery mildew, a devastating fungal disease. Similarly, gene editing has enabled the development of rice strains with enhanced resistance to bacterial blight, a disease that can lead to significant yield losses. In tomatoes, gene editing has been employed to confer resistance against the Tomato Yellow Leaf Curl Virus, which severely affects tomato production worldwide. These achievements underscore the potential of gene editing to create crops that are not only more resilient but also capable of maintaining high yields under disease pressure. However, the application of gene editing in crop disease resistance is not without challenges. One of the primary concerns is the regulatory landscape, which varies significantly across different countries. While some nations have embraced gene-edited crops, others have imposed strict regulations, treating them similarly to genetically modified organisms (GMOs). This inconsistency can hinder the global deployment of disease-resistant crops and create trade barriers. Additionally, there are concerns related to off-target effects, where unintended changes in the genome may occur, potentially leading to unintended consequences in the plant's growth or ecological interactions.

Long-Term Monitoring and Management of Genetically Modified Canola in Natural Environments: A 15-Year Study

The unintentional release of living modified organisms (LMOs) into natural environments poses potential ecological risks, particularly in terms of gene flow and biodiversity loss. Since 2009, the Ministry of Environment (MOE) in South Korea has conducted an extensive monitoring project to detect and manage LMOs, with a primary focus on LM canola. This study evaluates the outcomes of the LMO monitoring project over the past 15 years (2009–2023), analyzing the distribution, persistence, and management of LM canola across various environments. Our findings reveal that LM canola predominantly proliferates along roadsides, with occasional occurrences at festival and planting sites. Out of 10,571 monitored sites, 4326 suspicious samples were collected, with a significant increase observed in 2017, underscoring the ongoing risk of accidental releases. This study highlights the critical role of specific environments in the spread of LM canola, and assesses the effectiveness of post-management strategies in controlling these populations. The National Institute of Ecology (NIE) has developed and implemented advanced monitoring protocols and post-management systems tailored to the characteristics of the monitoring sites and the nature of the LMOs. These efforts have been effective in controlling the spread of LM canola, thereby helping to preserve the biodiversity of South Korea’s natural environments. In conclusion, the proactive and adaptive strategies employed by the NIE are essential for mitigating the ecological risks associated with LMOs. Our study emphasizes the importance of on-going vigilance and the continuous refinement of monitoring and management practices to safeguard biodiversity and ecosystems.

Advancements in genetically modified insect pest-resistant crops in India.

The review offers insights into the current state of research on insect pest-resistant GM crops and the regulations governing the cultivation of GM crops in India. India has a rich crop diversity of more than 160 major and minor crops through its diverse agroclimatic conditions. Insect pests alone cause around USD 36 billion in crop loss annually in India. The last two decades witnessed considerable progress in managing insect pests by adopting innovative techniques including transgenics. In research, significant advancement has been brought in insect pest-resistant transgenics in India since its inception in 2002. However, any events have not been endorsed owing to biosafety impediments, except Bt cotton reaching the commercial release stage. A landmark decision to exempt certain types of gene-edited plants from genetically modified organism (GMO) regulations offers great promise for developing novel insect-resistant crops in India. The article reviews the current research on insect pest-resistant transgenics and its regulations in India.

Nutritional value of a novel EMS-induced cell wall-defective strain of Saccharomyces cerevisiae yeast for Pacific oyster (Magallana gigas) juveniles

The difficulties and high costs of live microalgae production have driven the search for alternative diets in bivalve aquaculture. The baker's yeast (Saccharomyces cerevisiae) represents a promising, cost-effective, high-quality protein for aquaculture. However, its application is restricted by poor digestibility due to its thick, rigid cell wall. Previously, we observed that the deletion of mnn9 gene in S. cerevisiae, leading to defective mannan synthesis and increased β-glucan exposure, enhances growth and immune response in Magallana gigas oysters. Nevertheless, using ∆mnn9 in aquaculture is not feasible due to restrictions on genetically modified organisms (GMO) containing antibiotic-resistance genes. This study aimed to create a non-GMO cell-wall defective yeast mutant (JH40) via ethyl methanesulfonate (EMS) mutagenesis and evaluate its nutritional value for M. gigas juveniles. Phenotypical differences between wild-type S. cerevisiae (WT), ∆mnn9, and JH40 were characterized using fluorescein isothiocyanate (FITC)-labeled lectin analysis, microscopy, cell diameter measurements, growth curves, and osmosensitivity tests. The nutritional value of a microalgae-based diet (Chaetoceros muelleri:Tisochrysis lutea, 50:50 based on dry weight) and its 50% substitution with JH40 or WT was assessed over 21 days in M. gigas juveniles. A complementary test also explored higher substitution levels (63%, 75%, and 100%) of the based diet with JH40 and the addition of extra JH40 (+25 %) in the 63% and 75% replacement diets. Results indicated that JH40 forms clumps, grows slower than the WT, has a larger cell diameter, higher binding affinity to WGA and LEL lectins than the WT, and is highly sensitive to hypo-osmotic stress, which are characteristics of cell-wall defective yeasts. Oysters fed a diet with 50% JH40 substitution showed significantly higher growth compared to those fed the WT-containing diet. JH40 could replace 50% of the algal diet without significantly affecting oyster growth and survival. However, higher inclusion levels of JH40 (62 %, 75 %, and 100 %) did not achieve the oyster growth observed with the algae-based diet, even with additional yeast. These findings are attributed to the limited polyunsaturated fatty acid content in yeast cells. Further research on dosing and administration frequencies is needed to evaluate the immunostimulatory potential of JH40.

A comprehensive analysis and exploration of the recent developments in the utilization of genetically modified microorganisms for the remediation of hazardous dye pollutants

Innovative and effective remediation techniques are required with regard to growing environmental concerns on hazardous dye pollutants emitted by the textile industries. The review explores on the recent developments in utilization of genetically modified organisms (GMO) for the dye removal. Through improved capacities and specificities towards dye pollutants, genetically modified organisms present a potential strategy towards achieving Sustainable Development Goal (SDG) No. 6 of the UN, focused on clean water and sanitation. The key findings show that genetic engineering methods, including recombinant DNA (rDNA) and CRISPR-Cas9 technologies, can increase the capacity of bacteria to degrade, metabolize, or immobilize dye pollutants, improving degradation efficiency from around 7%–65%. The enzymatic processes are identified as the primary mechanisms involved in dye compound degradation by genetically engineered microbes. The review emphasizes the use of genetic approaches for dye degradation, co-cultivation with native microbial communities, optimizing operational parameters like pH, temperature, and nutrient availability, and omics technologies for a deeper understanding of metabolic networks and regulatory mechanisms with the aim of leading future genetic advancements in dye remediation. The review discusses the practical feasibility and environmental safety challenges of using genetically engineered microbes in sustainable dye remediation, highlighting limitations and future insights.

Event-specific loop-mediated isothermal amplification for living modified cotton MON88701, MON531, MON15985, MON88913, and COT102

Gossypium hirsutum L., commonly known as upland cotton, is cultivated globally for natural fiber, feed, and seed oil. To enhance agricultural productivity and quality, living modified (LM) cotton has been developed and utilized since the late 1990s. Due to environmental concerns, such as biodiversity risks associated with living modified organisms (LMOs), the Korean government, including the Ministry of Environment, has been conducting LMO natural environment monitoring and post-management projects. In this study, we developed a loop-mediated isothermal amplification (LAMP) assay to detect five specific LM cotton events (MON88701, MON531, MON15985, MON88913, and COT102), which were the most abundant volunteers observed from 2019 to 2021 in South Korea. The event-specific LAMP assays for the five LM cotton events were established with a 40-min reaction time using LAMP reaction buffer, Bst DNA polymerase, and event-specific primers. The limit of detection ranged from 0.01 to 1 ng/μL. The assays' specificity and sensitivity were validated through colorimetric changes, fluorescence intensity measurements, and conventional PCR. To demonstrate the practical application of the LAMP assays, we tested 22 LM cotton volunteers collected from the natural environment in 2021. Each LAMP assay event-specifically amplified the respective LM cotton volunteers. These results indicate that the developed LAMP assays are effective tools for the efficient management and detection of LM cotton in field surveys, supporting regulatory compliance and environmental monitoring efforts.

In-Vitro Growth of Transgenic Sengon Resulting From Co-Cultivation Using Agrobacterium Tumefaciens Bacteria

Sengon (Paraserianthes falcataria (L) Nielsen) is a forestry plant often developed for industrial purposes in Indonesia. Sengon is a fast-growing species and has a relatively short-cutting period. Sengon wood is widely used in the production of carpentry wood, plywood raw materials, and pulp. The purpose of this study was to optimize the transformation of the entC and pmsB genes into sengon plants using the embryo injury method through Agrobacterium tumefaciens as an intermediary and to determine the efficiency of the transformation using the injury to sengon embryos. This research was conducted from September 2022 to March 2023. The research was conducted at the Genetically Modified Organism (GMO) Tissue Culture Laboratory, Recombinant Protein (Microbe) Laboratory, and Greenhouse of the Genetic Engineering Center, National Research and Innovation Agency (BRIN) Cibinong, Bogor, Java West. The research procedure consisted of several steps, namely Preparation of MS Media, Preparation of LB Media, Preparation of Sengon Seed Infection Using Agrobacterium tumefaciens, Sterilization and Planting of Sengon Seeds, Acclimatization, and Molecular Analysis (including Sengon DNA isolation, PCR Amplification, and Electrophoresis). Transformation efficiency was calculated by the positive strain of hpt PCR bands compared to the number of transformed embryos and multiplied by 100%. In the results of the PCR analysis, it was found that one plant sample carried the hpt marker, which indicates the integration of the entC and pmsB genes. which when analyzed using transformation efficiency calculations, the data obtained for the entire transformation stage was 0.8%, where the success of the transformation obtained was still classified as low. Factors that cause the low success of transformation include the method of transformation, the growth phase of bacteria, genetic material, and culture aseptic conditions.