10 sel kompeten e coli teratas 2022

Microbiol J India. 2018 Des; 58 (4): 448–456. 2018 Dec; 58(4): 448–456.

Abstrak

Sistem transformasi hemat, hemat biaya menentukan keberhasilan kloning gen dan analisis fungsional. Di antara berbagai faktor yang mempengaruhi sistem transformasi ini, kemampuan kompetensi sel target adalah salah satu faktor terpenting. Kami menemukan peptida antimikroba lfcin-B dapat meningkatkan permeabilitas membran sel, dan sifat antibakteri mematikannya dapat dihambat oleh konsentrasi Ca2+ dan Mn2+ yang cukup tinggi. Dalam penelitian ini, kami menetapkan metode yang nyaman dan cepat (CRM) dengan menambahkan konsentrasi kecil (0,35 & nbsp; mg/L) dan konsentrasi MnCl2 yang cukup tinggi (50 & nbsp; mM) dan CaCl2 (30 & nbsp; mM) dalam buffer transformasi. Efisiensi transformasi sel E. & NBSP; coli (DH5α, JM109 dan TOP10) yang disiapkan oleh CRM sebanding dengan elektroporasi untuk transformasi plasmid (3,1 ± 0,3 × 109 CFU/μg). Tidak seperti sel kompeten yang disiapkan menggunakan metode kimia lainnya, yang diperoleh dengan menggunakan metode CRM sangat kompeten untuk menerima fragmen DNA ukuran yang lebih besar (> 5000 & NBSP; BP) ke dalam vektor plasmid. Sel-sel COLI E. & NBSP; yang kompeten yang disiapkan dengan metode CRM sangat berguna untuk sebagian besar percobaan transformasi efisiensi tinggi dalam kondisi laboratorium normal.

Kata kunci: biotransformasi, sel yang kompeten, efisiensi transformasi tinggi, kloning molekulerBiotransformation, Competent cells, High transformation efficiency, Molecular cloning

pengantar

Transformasi melibatkan memperkenalkan DNA eksogen ke dalam bakteri reseptor untuk menghasilkan sifat genetik baru. Ini adalah teknik paling mendasar dan penting dalam kloning molekuler, yang merupakan proses mengisolasi fragmen DNA target dan membuat banyak salinannya [1]. Ada dua jenis metode transformasi: kimia dan fisik [2, 3]. Metode transformasi fisik adalah elektroporasi. Ini memiliki efisiensi transformasi yang tinggi hingga 109-1010 transforman/μg DNA di E. & nbsp; coli [2]. Beberapa penelitian lain juga telah melaporkan transformasi efisiensi tinggi melalui elektroporasi di Agrobacterium tunrefaciers/rhizobium dan Bacillus brevis [4-7]. Namun, peralatan khusus yang mahal diperlukan untuk elektroporasi, dan tidak semua laboratorium dapat menyediakannya. Transformasi kimia sangat disambut dalam percobaan kloning molekuler karena sederhana dan murah. Metode kimia klasik adalah metode kalsium klorida (metode CaCl2) yang diterbitkan oleh Mandel dan Higa, yang masih merupakan pilihan umum untuk banyak laboratorium [8]. Peran Ca2+ dalam metode ini adalah untuk menghancurkan array lipid pada membran sel, dan membentuk kompleks dengan poli-hidroksibutrate dan fosfat poli-organik pada membran sel untuk memfasilitasi infiltrasi DNA eksogen, dan efisiensi transformasi adalah 105–106 DNA transforman/μg yang terbukti cukup untuk sebagian besar tujuan kloning [9, 10]. Meskipun metode kalsium klorida nyaman dan berulang, persiapan sel yang kompeten relatif memakan waktu dan membutuhkan daya yang cukup besar. Yang lebih penting adalah bahwa efisiensi transformasi jauh lebih rendah daripada elektroporasi, yang membuatnya tidak cocok untuk beberapa percobaan kloning molekuler seperti konstruksi perpustakaan cDNA kompleksitas tinggi dengan pengeluaran minimum mRNA [11]. Sejumlah besar penelitian telah melakukan upaya untuk membangun metode yang cepat dan efisien untuk persiapan sel E. & NBSP; coli yang kompeten dengan frekuensi transformasi yang sangat tinggi, yang memenuhi persyaratan percobaan kloning molekuler modern [3, 12-16] . Ada dua jalan untuk meningkatkan protokol persiapan, yang pertama menyederhanakan langkah -langkah dan waktu persiapan korslet dan yang kedua meningkatkan frekuensi transformasi sel yang kompeten secara kimia [11, 17-21]. Namun, tidak ada metode yang dikutip di atas dapat memberikan sel E. & NBSP; coli yang kompeten yang sangat berguna untuk menerima fragmen DNA target besar ke dalam vektor plasmid. Sel-sel semacam itu adalah dasar untuk hampir semua percobaan molekuler seperti analisis lokalisasi subseluler, uji dua-hibrida ragi, analisis immunoassay fluoresensi, dan faktor-faktor serupa.

Semakin banyak penelitian telah melaporkan bahwa peptida antimikroba dapat membunuh mikroorganisme dengan menghancurkan membran sel mikroorganisme, dan efek dari beberapa peptida antimikroba, seperti meningkatkan permeabilitas membran sel, tidak mematikan bagi E. & nbsp; coli [22-24] . Untuk alasan ini, kami meningkatkan protokol yang dijelaskan oleh Inoue [11], yang merupakan salah satu metode kimia terbaik saat ini tersedia, dengan menambahkan konsentrasi kecil Lfcin-B, peptida antimikroba yang ditemukan pada mamalia [25] untuk menetapkan metode untuk tersebut Persiapan sel -sel tersebut efisien dalam plasmid dan transformasi DNA target yang besar.

Materials and Methods

Bacterial Strains, Plasmids and Chemicals

Three strains of E. coli (DH5α, TOP10 and JM109) were used in this study to prepare competent cells. The commercial competent cells DH5α and JM109 were purchased from Takara Biomedical Technology (Beijing) Co., Ltd, commercial competent cells TOP10 were purchased from TIANGEN BIOTECH (Beijing) Co., Ltd. The pUC19 plasmids and restriction enzymes used in our laboratory were purchased from New England Biolabs (USA). The pGEM-Teasy vector were purchased from Promega Corporation, an affiliate of Promega (Beijing) Biotech Co., Ltd. The other chemicals used in this study were purchased from Sigma-Aldrich Co. LLC or Sinopharm Chemical Reagent Co., Ltd.

Media for Bacterial Growth

For LB fluid medium, 1% Bacto–Tryptone (10 g/L), 0.5% Bacto–Yeast Extract (5 g/L), 0.5% NaCl (5 g/L), adjust the pH to 7.5, autoclave to sterilize. For LB plates, 1.5% Bacto-agar (15 g/L) was added prior to autoclaving. For S.O.C. fluid medium, 2% Bacto–Tryptone (20 g/L), 0.5% Bacto–Yeast Extract (5 g/L), 0.05% NaCl (0.5 g/L), 2.5 mM KCl (0.186 g/L), 1 mM MgCl2 (0.95 g/L), 10 mM MgSO4 (1.2 g/L), 75 mM (13.6 g/L) glucose, adjust the pH to 7.0, autoclave to sterilize. For S.O.B fluid medium, 2% Bacto–Tryptone (20 g/L), 0.5% Bacto–Yeast Extract (5 g/L), 0.05% NaCl (0.5 g/L), 2.5 mM KCl (0.186 g/L), 1 mM MgCl2 (0.95 g/L), adjust the pH to 7.0, autoclave to sterilize.

The Competent Cell Preparation Methods and Transformation Methods

There were three methods for competent cell preparation in this study. One was CaCl2 method [8, 26], the second was Inoue method [11], and the last one was our improve method (CRM). All these three competent cells were used for heat shock transformation, the details for preparation and transformation were described as follow:

CaCl2 Method [8, 26]

The CaCl2 method using to prepare competent cells and transformation were both followed the protocols described by Sambrook [26].

Inoue Method [11]

The transformation buffer (TB) was made of 10 mM Pipes, 55 mM MnCl2, 15 mM CaC12 and 250 mM KCI, the pH was adjusted to 6.7 with KOH. And then, the solution was sterilized by filtration through a preripsed 0.45 µm filter unit and stored at 4 °C. All salts were added as solids. Frozen stock E. coli competent cells were thawed, streaked on an LB agar plate, and cultured overnight at 37 °C. The large (diameter 2–3 mm) colonies were isolated and inoculated to 250 mL of SOB medium in a 2-L flask, and grown to an OD6oo of 0.6 at 18 °C, with vigorous shaking (200–250 rpm). The flask was removed from the incubator and placed on ice for 10 min. The culture was transferred to a 500 mL erlenmeyer flasks and centrifuged at 3000 rpm for 10 min at 4 °C. Discard the supernatant and resuspend the pellet in 80 mL of ice-cold TB, and then incubated in an ice bath for 10 min, and then centrifuge as above. Discard the supernatant and resuspend the pellet gently in 20 mL of DMSO-TB buffer [final concentration of DMSO (m/v) was 7%]. After incubating in an ice bath for 10 min, the cell suspension was dispensed by 1–2 mL into tissue-culture cell-freezing tubes and immediately chilled by immersion in liquid nitrogen. The frozen competent cells were stored in − 78 °C or used for transformation immediately.

Usually, 1–5 µL plasmid (ligation product) was added into the competent cells for transformation, and then the cells were incubated in an ice bath for 30 min. They were then heat-shoked without agitation at 42 °C for 30 s and transferred to an ice bath. After 0.8 mL of SOC was added, the tubes were placed in a 37 °C incubator and shaken vigorously for 1 h. A desired portion of the mixture was poured on the LB plate with ampicillin (final concentration 100 µg/mL). Colonies were counted after overnight incubation at 37 °C.

CRM Method (Our Improved Method)

The transformation buffer (TB) was made of 10 mM Pipes, 50 mM MnCl2, 30 mM CaC12, 250 mM KCl and 0.35 mg/L LFcin-B, the pH was adjusted to 6.7 with KOH. The solution was also sterilized by filtration through a 0.45 µm filter unit and stored at 4 °C.

Streak a LB agar plate with E. coli cells from a frozen stock. Incubate the plate upside down at 37 °C until colonies appear. Inoculate one colony into 500 mL S.O.C. liquid medium in a 1 L flasks, incubate at 18 °C with shaking at 100 rpm overnight until the OD600 reaches 0.6. Incubate cells at ice for 10 min, and then centrifuge the cells at 2500 rpm for 10 min at 4 °C. Discard the supernatant and resuspend the pellet in 16 mL TB. Incubate cells at ice for 10 min, and then centrifuge as above. Discard the supernatant and resuspend the pellet in 8 mL TB, and then centrifuge the cells at 2500 rpm for 10 min at 4 °C. Discard the supernatant and resuspend the pellet in 4 mL DMSO-TB buffer [final concentration of DMSO (m/v) was 7%]. Incubate the cells at ice for 30 min. Aliquot 100 μL into individual 1.5 mL tubes. Frozen in liquid nitrogen immediately and stored at − 78 °C or used for transformation immediately.

Untuk transformasi, langkah pertama adalah campuran dengan lembut 1–5 & nbsp; μl plasmid (produk ligasi) dengan sel yang kompeten. Kedua, inkubasi campuran di atas es untuk 30 & nbsp; min segera. Campuran itu kemudian dipanaskan tanpa agitasi pada 42 & nbsp; ° C untuk 30 & nbsp; dipindahkan ke es untuk 2 & nbsp; menit segera. Setelah 500 & nbsp; μl medium cairan LB ditambahkan ke dalam campuran, mereka diinkubasi pada 37 & nbsp; ° C dengan pengocok pada 200 & nbsp; rpm untuk 1 & nbsp; h. Bagian campuran yang diinginkan dituangkan pada pelat LB dengan ampisilin (konsentrasi akhir 100 & nbsp; μg/ml). Koloni dihitung setelah inkubasi semalam pada 37 & nbsp; ° C.

Eksperimen elektroporasi, skrining bintik biru dan PCR bakteri individu dilakukan sesuai dengan protokol yang dijelaskan oleh Sambrook [26].

Perhitungan efisiensi transformasi

Efisiensi transformasi (TE) didefinisikan sebagai jumlah CFU (unit pembentukan koloni) yang diproduksi oleh 1 & nbsp; μg DNA plasmid, persamaan untuk menghitung jumlah CFU transforman (TC) adalah sebagai berikut:

TC (CFU) = TheneMerofbacteriacolonies × DilutionRatio × OriginalTransformationVolumeplated volume

Dan kemudian efisiensi transformasi (TE) dihitung sesuai dengan persamaan berikut:

TE (CFU/μg) = DNA Tcplasmid (μg)

Dalam penelitian ini menurut metode, rasio pengenceran untuk metode Inoue dan CRM adalah 5 × 106 kali, untuk metode CaCL2 adalah 1 × 106 kali. Rasio pengenceran untuk sel -sel kompeten komersial juga dihitung sebagai 500.000 kali dalam penelitian ini. Untuk perhitungan efisiensi transformasi, DNA plasmid 2 & nbsp; μl (500 & nbsp; ng/μl) ditambahkan dalam sel kompeten 100 & nbsp; μl, volume berlapis adalah 50 & nbsp; μl. Ada tiga pengulangan untuk setiap pengujian.

Analisis Permeabilitas membran sel

Uji NPN digunakan untuk mendeteksi permeabilitas membran luar E. coli [27]. Sel E. coli dalam fase logaritmik dalam medium LB (hingga kepadatan optik OD600 0,4) dikumpulkan dan diresuspensi dengan normal & nbsp; saline. Ke 1 & nbsp; ml volume bakteri dalam kuvet kuarsa, NPN (N-phenyl-1-naphthylamine) ditambahkan (konsentrasi akhir: 10 & nbsp; µm). Konsentrasi lfcin-B yang berbeda ditambahkan untuk membuat konsentrasi mereka 0, 0,1, 0,2, 0,3, 0,4 dan 0,5 & nbsp; mg/L. Spektrofotometer fluoresensi (PE LS45) digunakan untuk merekam fluoresensi setiap 30 & nbsp; min, panjang gelombang eksitasi dan emisi ditetapkan masing -masing pada 350 dan 429 & nbsp; nm. Tes kontrol dilakukan untuk memverifikasi bahwa fluoresensi yang ditingkatkan disebabkan oleh penyerapan NPN oleh bakteri.

Permeabilitas membran dalam E. coli ditentukan dengan mengukur pelepasan aktivitas β-galaktosidase ke dalam media kultur menggunakan ONPG (ortho-nitrophenyl-β-galactoside) sebagai substrat [27]. β-galactosidase adalah enzim E. coli yang diinduksi yang terletak di dalam membran sel bakteri dan mampu hidrolisis laktosa menjadi glukosa dan galaktosa. ONPG dapat digunakan untuk mendeteksi aktivitas β-galactosidase karena itu adalah substrat kromogenik untuk β-galactosidase. Jika permeabilitas membran sel berubah, ONPG dapat masuk ke dalam sitoplasma dan mengkatalisasi β-galactosidase untuk menghasilkan produk kuning. Puncak penyerapan maksimum dari produk kuning ini adalah 415 & nbsp; nm [27]. Oleh karena itu, ONPG dipilih sebagai substrat untuk reaksi, dan permeabilitas membran bagian dalam E. coli ditentukan dengan menggunakan aktivitas β-galactosidase. Dalam penelitian ini, sel E. coli dalam fase logaritmik dalam medium LB yang mengandung 2% laktosa (hingga kepadatan optik OD600 0,4) dikumpulkan dan diresuspensi dengan normal & nbsp; saline juga. Suspensi sel E. coli (200 & nbsp; μl) dipipet ke dalam sumur pelat mikrotiter standar diikuti dengan menambahkan ONPG (konsentrasi akhir: 1.5 & nbsp; μm). Konsentrasi lfcin-B yang berbeda ditambahkan di setiap sumur untuk membuat konsentrasi mereka 0, 0,1, 0,2, 0,3, 0,4 dan 0,5 & nbsp; mg/L. Sedikit getaran pada 37 & nbsp; ° C dan produksi o-nitrofenol dari waktu ke waktu dipantau dengan spektrofotometer pada 415 & nbsp; nm.

Ekspresi procaryotic dan pemurnian antimikroba peptida laktoferrisin B (lfcin B)

Lfcin B yang digunakan dalam penelitian ini diperoleh dengan ekspresi prokariotik dan pemurnian di laboratorium kami menurut protokol Feng [28]. Kemurnian dan aktivitas antimikroba Lfcin B yang diproduksi di laboratorium kami sama dengan Lfcin B yang dimurnikan dari pencernaan sapi oleh Laboratorium Pusat Ilmu Pangan & Teknologi (Universitas Pertanian Huazhong, Wuhan, Republik Rakyat Tiongkok).

Hasil

Konsentrasi tinggi MnCl2 dan CaCl2 disertai dengan konsentrasi kecil Lfcin-B meningkatkan kompetensi DH5α

Competent cells are those that have had their cell membranes altered to render it easier to bring foreign DNA inside. Therefore, the permeability of cell membranes is the most important and fundamental condition for competent cell with high transformation efficiency. To improve the transformation efficiency of competent cells, we began to optimize the Inoue method by increasing the permeability of cell membranes. Because the E. coli DH5α strain is the most common competent cell used in DNA cloning experiments, we selected this strain to examine the permeability of the inner and outer layers of the cell membrane. According to the research on antimicrobial peptides which can affect the cell membranes of microorganism such as E. coli [22, 23], we first added three common antimicrobial peptides in LB medium at small concentrations (0.5 mg/mL) to assess their effects on the survival of E. coli DH5α strains. The results showed that the E. coli DH5α clones only grew LB medium and in LB with LFcin-B, but there were significant differences (P < 0.05) between the number of clones. LB medium had more than twice as many as LB-LFcin-B+ (195–213) versus 525–567, Fig. 1a). This indicates that the E. coli DH5α strain could grow on small concentrations (< 0.5 mg/L) of LB-LFcin-B+ medium, which means that LFcin-B can be added to the transformation buffer used to prepare competent cells to obtain cells with strong competence and high permeability. We then examined the variations in permeability of cell membrane caused by different concentrations of LFcin-B. The results of inner and outer cell membrane permeability both showed that the more LFcin-B present, the greater the permeability of the inner and outer cell membrane. We also found the influence on permeability of different concentration can be divided into three groups based on their differences from controls (without LFcin-B): the small effect (P > 0.05) of trace concentrations of LFcin-B (0.1–0.2 mg/L), the significant effect (P < 0.05) caused by small concentrations (0.3–0.4 mg/L) and the huge effect (P < 0.01) caused by normal concentrations (> 0.5 mg/L, Fig. 1b). To establish the influence of LFcin-B on the competence of E. coli DH5α strain, we added the same gradient concentration of LFcin-B into Inoue transformation buffer, and we found that the transformation efficiency of DH5α decreased as the concentration of LFcin-B increased (Fig. 1c). The low transformation efficiency may have been caused by the antibacterial properties of LFcin-B [25]. In the course of next investigation, we found that moderately high concentration of MnCl2 (50 mM) and CaCl2 (30 mM) with slight concentration of LFcin-B (0.35 mg/L) can be stimulatory to transformation by increasing the permeability of cell membrane (Fig. 1d). This may be due to the antibacterial property of LFcin-B can be inhibitory by high concentration of Ca2+ and Mn2+ without losing the increase influence of LFcin-B on DH5α cell membrane, as the mechanism about the antibacterial property of LFcin-B is complex and there remain many unresolved issues [22, 25]. The above observation about the increase in the competence of E. coli in the presence of LFcin-B, Ca2+, and Mn2+ at moderate concentrations suggested that it may improve the preparation of competent cells.

The effect of LFcin-B on the competence of DH5α. a The survival of DH5α grow on LB medium plate with different imicrobial peptides. Three common imicrobial peptides Cecropin A, PR-39 and LFcin-B were added in LB medium plate in normal concentration (0.5 mg/mL). LB medium plate was as control. DH5α strains were grow in these plates overnight at 37°C and then observed, calculated and photographed. b LFcin-B increase the permeability of inner and outer cell membrane determined by NPN assay and ONPG assay. c The increase of LFcin-B decreased the transformation efficiency of DH5α. d Different concentration of MnCl2 and CaCl2 effect the antibacterial property of LFcin-B. Slight concentration of LFcin-B (0.35 mg/L) were added in Inoue method transformation buffer with gradient concentration of MnCl2 and CaCl2, and then calculated the transformation efficiency of DH5α competent cells made by these TB with Inoue method. Each data had three repeats and the bar indicated the SE

The CRM Method Can Produce Better-Quality Competent Cells than Previously Reported Methods

In molecular cloning experiments, transformation efficiency is commonly used as an index to assess the competence of competent cells. CaCl2 and Inoue methods were chosen to compare with CRM method in terms of the quality of competent cells because CaCl2 method was the most widely used chemical method of preparing competent cells in laboratories and the Inoue method can produce stable competent cells in a highly efficient manner [16]. In the first place, the transformation efficiency for pUC19 of three common competent E. coli strains (DH5α, JM109, and TOP10) separately prepared by CaCl2 method, Inoue method and CRM method were calculated and compared. Results showed that the transformation efficiency of all three E. coli competent strains (DH5α, JM109 and TOP10) in this study were similar. The transformation efficiency of CRM method was 3.2–3.5 × 109 cfu/µg for DH5α, 1.8–2.2 × 109 cfu/µg for JM109 and 3.4–3.7 × 109 cfu/µg for TOP10, which were all significantly higher (P < 0.05) than those of the Inoue method (1.9–2.8 × 109 cfu/µg for DH5α, 0.8–1.2 × 109 cfu/µg for JM109 and 2.7–3.1 × 109 cfu/µg for TOP10), and very significantly higher (P < 0.01) than those associated with the CaCl2 method (3.0–3.7 × 107 cfu/µg for DH5α, 3.9–4.3 × 107 cfu/µg for JM109 and 1.6–2.3 × 107 cfu/µg for TOP10). These results indicated that our improved method (CRM method) can be used to make at least three kinds of E. coli competent strains with higher transformation efficiency than the other widely used chemical method (Inoue method and CaCl2 method), and this may be true of other E. coli strains. Electro-transformation is a non-chemical method widely used in molecular cloning experiments due to its high transformation efficiency [2]. We also compared these methods. It gave a transformation efficiency of (2.9–3.6 × 109 cfu/µg for DH5α, 1.7–2.3 × 109 cfu/µg for JM109 and 3.1–3.6 × 109 cfu/µg for TOP10) of pUC19, a value comparable to the frequency (P > 0.5) of CRM method (data not shown). However, electroporation requires special equipment that many laboratories cannot provide. These results indicated that the CRM method can produce competent E. coli cells with high transformation efficiency (1.8–3.7 109 cfu/µg), much higher than other chemical transformation and comparable to electro-transformation, indicating that it is sufficient for the genetic transformation necessary to create high-quality competent cells (Fig. 2).

The transformation efficiency (TE) of three E. coli competent cells (DH5α, TOP10 and JM109) made by different methods. Bars represent standard error (n = 3). One and double asterisks above the columns indicated significant difference at p < 0.05 and p < 0.01 separately

In modern molecular biology, increasing numbers of experiments require insertion of target DNA fragments into specific plasmids using PCR, restriction enzyme digestion, ligation reactions, and transformation. The larger the target DNA fragment, the fewer positive clones are obtained. To investigate the competent cells prepared by CRM method has better ability to transfer large target DNA fragments than other methods. A 8105 bp ligation product which connected a 5100 bp gene fragment (AT3G52250.1) with a 3015 bp double digestion pGEM-Teasy vector fragment was transferred using different competent cells prepared using different methods. The transformants were selected on LB agar containing IPTG, X-gal, and ampicillin. Blue and white colonies were observed after overnight incubation. White colonies indicate positive transformed colonies with target DNA fragments and blue colonies indicate negative colonies that do not contain the target DNA fragment. Results show that, among three DH5α strains prepared using different methods, only the CRM method gave positive results. No transformants appeared in CaCl2 method plate, and there were only negative colonies (blue) in Inoue method plate (Fig. 3a). As no positive colonies were observed on the Inoue and CaCl2 method plates, we were not able to perform statistical analysis of these data. Two white transformants from CRM method plate and two blue transformants were randomly chosen to extract plasmids. These plasmids were subjected to PCR and enzyme digestion for confirmation of target DNA insertion in plasmids. The result of PCR and enzyme digestion both confirmed that the target DNA were successfully inserted into plasmid only by competent cell using CRM method (Fig. 3b, c). Further sequence reaction also proved this (data not shown). These results indicated that the competent cells prepared using the CRM method was best for inserting larger (> 5000 bp) target DNA fragments into plasmid vector than other competent cells were. This may be because of the increased cell membrane permeability attributable to addition of LFcin-B. Results indicate that the CRM method can not only produce competent E. coli cells with better transformation efficiency than other chemical methods, comparable to the electro-transformation method, and it can also produce highly competent E. coli cells, which are extremely efficient for incorporating larger DNA fragments into plasmid vectors.

Inserting larger size of target DNA fragment into plasmids by different E. coli competent cells. a Blue-white spot screening experiment of a 8105 bp ligation product using different E. coli competent cells. White colonies indicate positive transformed colonies with target DNA fragment and blue one indicate negative colonies without target DNA fragment. b PCR assay for confirmation of target DNA insertion in plasmid. Line 1 and 2 were the gel electrophoresis results of CRM method PCR products, line 3 was the marker, line 4 and 5 were the gel electrophoresis results of Inoue method PCR products. c Enzyme digestion assay for confirmation of target DNA insertion in plasmid. Line 1 and 6 were marker. Line 2 and 3 were the gel electrophoresis results of CRM method enzyme digestion products, line 4 and 5 were the gel electrophoresis results of Inoue method enzyme digestion products

Discussion

Cloning PCR products into plasmid vectors is a common and necessary upstream application for most modern molecular cloning experiments. Transformation efficiency is very important because it can directly affect the success of all the follow-up assays, and it can be affected by factors such as the quality of competent cells. Electroporation, which has high transformation efficiency requires special and expensive equipment [9, 10], so the establishment of a convenient and rapid chemical method of transformation for all E. coli bacterial strains and large DNA fragments is necessary and important to the development of modern molecular biology. As the increasing reports on the non-lethal effect of some antimicrobial peptide by increasing the permeability of cell membrane [22, 23], we here improved upon the Inoue method by adding various common antimicrobial peptides found in mammals [22]. We found one antimicrobial peptide, LFcin-B, to have a non-lethal effect on E. coli DH5α strains. Through repeated attempts, we found that the low transformation efficiency caused by LFcin-B’s antibacterial property [25] can be increased by adding moderate high concentration of MnCl2 (50 mM) and CaCl2 (30 mM). This may be because the antibacterial properties of LFcin-B can be inhibited by high concentrations of Ca2+ and Mn2+ without losing the increasing permeability of the DH5α cell membrane [22, 25]. Although the mechanism underlying the antibacterial properties of LFcin-B is complex and still remain unclear, the observation can utilized to obtain competent cells with high competence which is urgently necessary in modern molecular cloning experiments because the permeability of the cell membrane directly influences the competence of E. coli cells. We are the first team to prepare highly competent cells by adding moderate concentrations of LFcin-B. Further assays indicated that CRM method with its small concentration of LFcin-B (0.35 mg/L) with moderately high concentrations of MnCl2 (50 mM) and CaCl2 (30 mM) can also increase the transformation efficiency of other two common E. coli strains, JMI09 and TOP10. These results indicated that CRM method can applied to produce high quality competent cells of most common E. coli cells which means this method can be widely used. Compared with other chemical methods (CaCl2 and Inoue), CRM can produce competent E. coli cells with much higher transformation efficiency than those produced with other methods (1.8–3.7 × 109 cfu/µg), comparable to electro-transformation. The best-known protocol for preparing competent E. coli cells and chemical transformation is the CaCl2 method published by Mandel and Higa [8]. This method is still widely used in many laboratories due to its convenience. Another good chemical methods was described by Inoue [11] because its transformation efficiency has been carefully optimized through a great number of studies [3, 15, 29–31]. The basic steps of this technique have undergone a few modifications for optimizing the efficiency of transformation, the majority of these efforts were indeed able to enhance the transformation efficiencies but the increase depended on suitable smaller DNA and differences among E. coli bacterial strains [10, 32, 33]. Unlike this methods, CRM method can increase the transformation efficiencies at least three common strains of E. coli cells, which strongly suggests it may be applied in most E. coli cells, and the huge increased transformation efficiencies exceed the maximum previous report. Moreover, the competent cells prepared by CRM method not only have high transformation efficiency, which is sufficient for modern molecular experiments but also have an especially good ability to insert larger DNA fragments into plasmid vectors, which Inoue and other transformation methods do not tend to offer. With the increasing development of genetic recombination technology, the most convenient and effective pathway to producing LFcin-B protein has been expression recombinant target protein in vitro [34]. At present, there are four major systems for production recombinant protein in vitro. These are the prokaryotic protein expression system, mammalian cell protein expression system, yeast protein expression system, and insect cell protein expression system [35]. According to previous reports, it is possible to produce good-quality LFcin-B protein via prokaryotic protein expression system and yeast protein expression system [28, 36–39]. Producing recombinant LFcin-B protein through prokaryotic protein expression systems is more welcome both in research and industrial production because E. coli has a clear genetic background and strong tolerance to many proteins, which means high levels of expression of the target proteins can be induced under appropriate conditions [28, 38]. Producing LFcin-B protein using the prokaryotic expression system offers convenient operation, low cost, fast speed, and large-scale, high-quality expression. Adding small concentration of LFcin-B is not expensive or troublesome, and the cost of CRM method in both time and money are similar to those of other chemical methods, but the transformation efficiencies and level of competence of competent cells can be significantly increased by the CRM method.

Sebagai kesimpulan, kami telah menggambarkan protokol untuk mempersiapkan sel E. & NBSP; coli yang paling cocok untuk transformasi plasmid yang efisien dan penyisipan fragmen DNA yang lebih besar ke dalam vektor plasmid dalam penelitian ini. Kloning produk PCR ke dalam vektor plasmid adalah aplikasi hulu yang umum dari banyak penelitian molekuler modern seperti Co-IP dan BIFC [26]. Metode kami sederhana, dapat diandalkan, dan sangat dapat direproduksi. Ini bekerja untuk setidaknya tiga strain E. & nbsp; coli (DH5α, JMI09, dan TOP10), dan mungkin strain E. & nbsp; coli lainnya. Hasil yang dilaporkan di sini dapat berguna untuk berbagai studi biologis.

Ucapan Terima Kasih

Studi ini didukung oleh Natural Science Foundation of Hubei Province (hibah No.2016CFB630), Penelitian Dasar untuk Proyek Aplikasi Wuhan (Grant No. 2015011701011595) dan National Science Foundation of China (NSFC31701100, NSFC31600981, NSFC316001). Kami juga ingin mengucapkan terima kasih kepada Letpub (www.letpub.com) karena memberikan bantuan linguistik selama persiapan naskah ini.

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