Introduksi Vektor Ekspresi pGWB5-SOD pada Bakteri Escherichia coli DH5α dengan Metode Kejut Panas
Keywords:
DH5α, Escherichia coli, Kejut Panas, Vektor EkspresiAbstract
Transformasi bakteri adalah proses gen asing dimasukkan ke dalam sel bakteri dan mengekspresi gen tersebut. Proses transformasi bakteri melibatkan banyak metode, salah satunya adalah metode kejut panas. Metode kejut panas digunakan untuk meningkatkan kompetensi sel bakteri dan permeabilitas membrannya. Sehingga, gen asing dapat masuk ke dalam sel bakteri. Salah satu bakteri yang dapat digunakan untuk transformasi adalah Escherichia coli. Bakteri Escherichia coli secara umum digunakan dalam biologi molekuler sebagai bakteri uji. Bakteri Escherichia coli diketahui memiliki banyak strain, salah satunya adalah DH5α. Bakteri Escherichia coli DH5α merupakan strain yang mudah menerima gen asing. Karena mudah dibuat menjadi sel kompeten, tidak ada plasmid F, tidak ada enzim endonuklease 1 (mutasi gen endA1), dan memiliki penanda seleksi berupa resistensi antibiotik ampisilin. Transformasi bakteri Escherichia coli DH5α dengan vektor ekspresi pGWB5 mengandung gen SOD belum diteliti dan belum diketahui keberhasilannya. Sehingga, tujuan penelitian dilakukan untuk memasukkan vektor ekspresi pGWB5-SOD ke dalam bakteri Escherichia coli DH5α melalui metode kejut panas dan menganalisis ekspresinya melalui amplifikasi PCR koloni sebagai konfirmasi keberhasilan transformasi. Metode penelitian yang digunkan adalah metode kejut panas dengan vektor ekspresi pGWB5-SOD dan bakteri Escherichia coli DH5α. Transformasi bakteri dilakukan sebanyak 10 ulangan teknis pada kultur bakteri dan amplifikasi gen SOD dengan PCR. Hasil penelitian menunjukkan vektor ekspresi pGWB5-MmSOD berhasil masuk ke dalam bakteri Escherichia coli DH5α dan terjadi transformasi. Keberhasilan ini diperoleh dari koloni bakteri yang tumbuh dan muncul pita setelah amplifikasi dengan PCR.
References
Abulmagd S, Khattab AEA, Zedan H. (2022). Expression of full and fragment-B of diphtheria toxin genes in Escherichia coli for generating of recombinant diphtheria vaccines. Clin Exp Vaccine Res. 11(1):12–29. doi: 10.7774/cevr.2022.11.1.12.
Alam ST, Le TAN, Park JS, Kwon HC, Kang K. (2019). Antimicrobial biophotonic treatment of ampicillin-resistant Pseudomonas aeruginosa with hypericin and ampicillin cotreatment followed by orange light. Pharmaceutics. 11(12):641. doi: 10.3390/pharmaceutics11120641.
Arnak R, Altun B, Tosato V, Bruschi CV. (2016). Multiple antibiotic resistance plasmids allow scalable, PCR-mediated DNA manipulation and near-zero background cloning. Food Technol Biotechnol. 54(3):257–265. doi: 10.17113/ftb.54.03.16.4230.
Asif A, Mohsin H, Tanvir R, Rehman Y. (2017). Revisiting the mechanisms involved in calcium chloride induced bacterial transformation. Front Microbiol. 8:2169. doi: 10.3389/fmicb.2017.02169.
Bergkessel M, Guthrie C. (2013). Colony PCR. Methods Enzymol. 529:299–309. doi: 10.1016/B978-0-12-418687-3.00025-2.
Bhattacharjee D, Sorg JA. (2020). Factors and Conditions That Impact Electroporation of Clostridioides difficile Strains. mSphere. 5(2):e00941-19. doi: 10.1128/mSphere.00941-19.
Chan WT, Verma CS, Lane DP, Gan SK. (2013). A comparison and optimization of methods and factors affecting the transformation of Escherichia coli. Biosci Rep. 33(6):e00086. doi: 10.1042/BSR20130098.
Chang AY, Chau VWY, Landas JA, Pang Y. (2017). Preparation of calcium competent Escherichia coli and heat-shock transformation. JEMI Methods. 1:22–25.
Charpentier X, Polard P, Claverys JP. (2012). Induction of competence for genetic transformation by antibiotics: convergent evolution of stress responses in distant bacterial species lacking SOS? Current Opinion in Microbiology. 15(5):570–576. doi: 10.1016/j.mib.2012.08.001.
Cieślińska A, Kostyra E, Fiedorowicz E, Bukało M, Kocbach B, Matysiewicz M. (2014). Identifying stability of polymerase in master mixes used in PCR and repair possibilities for the degraded reagents. Polish Annals of Medicine. 21(2):82–85. doi: 10.1016/j.poamed.2014.07.009.
Contreras SUJ, Gardner CM. (2022). Environmental fate and behaviour of antibiotic resistance genes and small interference RNAs released from genetically modified crops. Journal of Applied Microbiology. 133(5):2877–2892. doi: 10.1111/jam.15741.
Datey A, Subburaj J, Gopalan J, Chakravortty D. (2017). Mechanism of transformation in Mycobacteria using a novel shockwave assisted technique driven by in-situ generated oxyhydrogen. Sci Rep. 7:8645. doi: 10.1038/s41598-017-08542-5.
Deng X, Yang W, Shao Z, Zhao Y. (2021). Genetically modified bacteria for targeted phototherapy of tumor. Biomaterials. 272:120809. doi: 10.1016/j.biomaterials.2021.120809.
Domenech A, Slager J, Veening JW. (2018). Antibiotic-induced cell chaining triggers pneumococcal competence by reshaping quorum sensing to autocrine-like signaling. Cell Reports. 25:2390–2400. doi: 10.1016/j.celrep.2018.11.007.
Dwiputri AW, Ratnayani K, Yowani SC. (2013). Amplication of 0.7kb fragment katg gene from clinical multi drug resistant Tuberculosis isolate in bali. Indonesia Journal of Biomedical Science. 7(2):69072.
Ghazaei C. (2019). Investigating the effect of thermal shock factors and different cell treatments on bacterial transformation efficiency. J Bas Res Med Sci. 6(1):49–56.
Hamed AA, Khedr M, Abdelraof M. (2020). Activation of LacZ gene in Escherichia coli DH5α via α-complementation mechanism for β-galactosidase production and its biochemical characterizations. J Genet Eng Biotechnol. 18(1):80. doi: 10.1186/s43141-020-00096-w.
Huang M, Liu M, Huang L, Wang M, Jia R, Zhu D, Chen S, Zhao X, Zhang S, Gao Q, Zhang L, Cheng A. (2021). The activation and limitation of the bacterial natural transformation system: The function in genome evolution and stability. Microbiological Research. 252:126856. doi: 10.1016/j.micres.2021.126856.
Imamuddin H. (2011). Uji resistensi terhadap HgCl yang diisolasi dari tanah penambangan emas di pongkor, jawa barat. Berita Biologi. 10(4):424–430.
Kalle E, Kubista M, Rensing C. (2014). Multi-template polymerase chain reaction. Biomol Detect Quantif. 2:11–29. doi: 10.1016/j.bdq.2014.11.002.
Kant R, Geeta B, Vinay KP, Anurup G, Deepak S, Monalisha N, Keerti M, Ankur G, Keshab G, Shubhra G, Gurunath R, Shantanu B. (2019). Synchronized electromechanical shock wave-induced bacterial transformation. ACS Omega. 4(5):8512–8521. doi: 10.1021/acsomega.9b00202.
Kasarjian JK, Iida M, Ryu J. (2003). New restriction enzymes discovered from Escherichia coli clinical strains using a plasmid transformation method. Nucleic Acids Res. 31(5):e22. doi: 10.1093/nar/gng022.
Liu J, Chang W, Pan L, Liu X, Su L, Zhang W, Li Q, Zheng Y. (2018). An improved method of preparing high efficiency transformation Escherichia coli with both plasmids and larger DNA fragments. Indian J Microbiol. 58(4):448–456. doi: 10.1007/s12088-018-0743-z.
Liu X, Liu L, Wang Y, Wang X, Ma Y, Li Y. (2014). The study on the factors affecting transformation efficiency of E. coli competent cells. Pak J Pharm Sci. 27(3):679–684.
Mizuno M, Matsuzaki T, Ozeki N, Katano H, Koga H, Takebe T, Yoshikawa HY, Sekiya I. (2022). Cell membrane fluidity and ROS resistance define DMSO tolerance of cryopreserved synovial MSCs and HUVECs. Stem Cell Res Ther. 13:177. doi: 10.1186/s13287-022-02850-y.
Moo-Young M. (2011). Comprehensive biotechnology. Amsterdam, Netherlands: Elsevier.
Mugwanda K, Hamese S, Van Zyl WF, Prinsloo E, Du Plessis M, Dicks LMT, Thimiri Govinda Raj DB. (2023). Recent advances in genetic tools for engineering probiotic lactic acid bacteria. Biosci Rep. 43(1):BSR20211299. doi: 10.1042/BSR20211299.
Musawira, Suharsono, Miftahudin, Tjahjoleksono A. (2022). Establishment of transgenic potato cultivar IPB CP1 plants containing gene encoding for superoxide dismutase to increase the abiotic stress tolerance. Indonesian Journal of Biotechnology. 27(3):118–125. doi: 10.22146/ijbiotech.68040.
Nouemssi SB, Ghribi M, Beauchemin R, Meddeb-Mouelhi F, Germain H, Desgagné-Penix I. (2020). Rapid and efficient colony-PCR for high throughput screening of genetically transformed Chlamydomonas reinhardtii. Life. 10(9):186. doi: 10.3390/life10090186.
O’Brien S, Baumgartner M, Hall AR. (2021). Species interactions drive the spread of ampicillin resistance in human-associated gut microbiota. Evol Med Public Health. 9(1):256–266. doi: 10.1093/emph/eoab020.
Pandey A, Teixeira JAC. (2017). Current developments in biotechnology and bioengineering: foundations of biotechnology and bioengineering. Amsterdam, Netherlands: Elsevier.
Patigu RF, Wijayanti P, Sebastian A, Purwestri YA. (2021). Optimization of heat shock temperature and time on the transformation of pRGEB32 into Escherichia coli DH5α. Jurnal Biologi Tropis. 21(3):632–640.
Pratiwi R. (2012). Perakitan vektor ekspresi protein G subunit α dari kedelai (Glycine max) kultivar Slamet [Tesis]. Bogor: Institut Pertanian Bogor.
Prout J, Tian M, Palladino A, Wright J, Thompson JF. (2023). LNA blockers for improved amplification selectivity. Sci Rep. 13(1):4858. doi: 10.1038/s41598-023-31871-7.
Putra LAG, Christianus JY, Nabila IN, Mochammad HRF, Jessica RY. (2020). A review of the development of polymerase chain reaction technique and its uses in scientific field. Stannum: Jurnal Sains dan Terapan Kimia. 2(1):17–30. doi: 10.33019/jstk.v2i1.1619.
Raharjo TJ, Surajiman. (2017). PCR primer specific CaMV 35S promoter to detect transgenic soybean in Indonesia commercial soybean and tempeh. Indones J Chem. 17(3):415–421. doi: 10.22146/ijc.24163.
Rahimzadeh M, Sadeghizadeh M, Najafi F, Arab S, Mobasheri H. (2016). Impact of heat shock step on bacterial transformation efficiency. Mol Biol Res Commun. 5(4):257–261.
Rotinsulu SH, Fatimawali, Tallei TE. (2019). Transformasi plasmid yang mengandung gen merB pada bakteri Escherichia coli TOP-10. Pharmacon. 8(2):290–297.
Setiari N, Purwantoro A, Moeljopawiro S, Semiarti E. (2018). Micropropagation of Dendrobium phalaenopsis orchid through overexpression of embryo gene AtRKD4. AGRIVITA Journal of Agricultural Science. 40(2):284–294. doi: 10.17503/agrivita.v40i2.1690.
Silalahi D, Wirawan IGP, Sasadara MMV. (2021). Optimization of annealing temperature for amplification of Ehoscn01a locus in pranajiwa (Euchresta horsfieldii) plant. IOP Conf Series: Earth and Environmental Science. 913:012059. doi: 10.1088/1755-1315/913/1/012059.
Sirajuddin SA, Sundram S. (2020). Evaluation of two transformation protocols and screening of positive plasmid into Bacillus cereus EB2. Braz J Microbiol. 51(3):919–929. doi: 10.1007/s42770-020-00241-0.
Taiwo SS. (2019). Overview of molecular diagnosis in medical microbiology. PAJOLS. 1:25–32. doi: 10.36108/pajols/8102/10(0150).
Thompson M, Yildirim S. (2019). Transformation of Acinetobacter baumannii: electroporation. Methods Mol Biol. 1946:69–74. doi: 10.1007/978-1-4939-9118-1_7.
Verma K, Dalal J, Sharma S. (2014). Scientific concepts of polymerase chain reaction (PCR). IJPSR. 5(8):3086–3095.
Voigt C. (2011). Methods in enzymology volume 498. Amsterdam, Netherlands: Elsevier.
Walch G, Knapp M, Rainer G, Peintner U. (2016). Colony-PCR is a rapid method for DNA amplification of Hyphomycetes. J Fungi (Basel). 2(2):12. doi: 10.3390/jof2020012.
Wang Y, Wang X, Yu L, Tian Y, Li S, Leng F, Ma J, Chen J. (2020). Effects of Sr2+ on the preparation of Escherichia coli DH5α competent cells and plasmid transformation. PeerJ. 8:e9480. doi: 10.7717/peerj.9480.
Wong G, Wong I, Chan K, Hsieh Y, Wong S. (2015). A rapid and low-cost PCR thermal cycler for low resource settings. PLoS One. 10(7):e0131701. doi: 10.1371/journal.pone.0131701.
Young JL, Dean DA. (2014). Electroporation-mediated gene delivery. Adv Genet. 89:49–88. doi: 10.1016/bs.adgen.2014.10.003.
Zhang Z, Kermekchiev MB, Barnes WM. (2010). Direct DNA amplification from crude clinical samples using a PCR enhancer cocktail and novel mutants of Taq. J Mol Diagn. 12(2):152–161. doi: 10.2353/jmoldx.2010.090070.
Zhou J, Li X, Xia J, Wen Y, Zhou J, Yu Z, Tian B. (2018). The role of temperature and bivalent ions in preparing competent Escherichia coli. 3 Biotech. 8(5):222. doi: 10.1007/s13205-018-1243-x.
Downloads
Published
Issue
Section
License
Copyright (c) 2024 Rinda Mulmeyda, Imam Safir Alwan Nurza
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
