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    In this paper we describe an electroporation protocol for Neisseria meningitidis.

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    Electroporation of Neisseria meningitidis with plasmid DNA

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    In this paper we describe an electroporation protocol for Neisseria meningitidis.

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    © All Rights Reserved
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    12 views3 pages

    Electroporation of Neisseria Meningitidis With Plasmid DNA

    Uploaded by

    Alejandro Martin
    In this paper we describe an electroporation protocol for Neisseria meningitidis.

    Copyright:

    © All Rights Reserved
    Download as PDF or read online from Scribd
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    ELECTROPORATION OF Neisseria meningitidis WITH PLASMID DNA 190 Duefas, = Rolando Pajén, Maité Delgado, Erick C Nogueiras, Alejandro Martin Centro de Ingenieria Genética y Biotecnologia. AP 6162, CP 10600, Ciudod de ta Habana, Cuba. Fax: (53-7} 218070; E-mail; ricardo.sila@cigb.edu.cy ABSTRACT In this paper we describe an electroporation protocol for Neisseria meningitidis. Plasmid vectors up to 12.5 kb, containing a selection marker, flanked by ns. When nonpil 272 mM; HEPES 1 mM pl 7.2}, ware mixed wth 200 rg of plasmid DNA and a single pulse of 1 ‘ond 25 mF was apy genomic regions, were constructed and used to select electroporation ion butter (glycerol 15%; MaCl, 1 mM; Sucrose $ kV/em, 400 2 (in BioRad Gene Pulser I), transformation efficiencies up to 6.5 x 10* transformants/ig ted B985 cells, suspended in electropora cand survival rates of up fo 80% were reached. The generation with this method, of an IpdA neisserial knock-out ‘mutant, is also shown. To date t ‘the first electroporation report in meningococc Keywords: electroporation, N. meningitidis, transformation efficiency Biotecnologia Aplicada 1998;15:247-249 RESUMEN En este arficulo describimos vn protocolo de electroporacién para Neisseria meningitidis. Varios plasmidios de hasta 12,5 kb, contenlendo regiones genémicas interrumpidas por un marcador de seleccién, fueron construidos y usados para establecer las condiciones de electroporacién. Cuando se mezclaron células de 8385 no piliadas, suspendidas en el tam} con 200 ng de ADI transformantes/} nde electroporacién (15% glicerol; 1 mM MgCl,; 272 mM sacarosa; 1 mM HEPES; pH 7,2), plasmidico, se obtuvieron eficiencias de transformacién que aleanzoron 6,5 x 10° on porcientos de supervivencia alrededor de 80. También se muestra la generacién, a través de este método, de un mutante de N. menigitidis para el gen IpdA. Este constituye el primer reporte describiendo la técnica de electraporacién aplicada a meningococo. Palabras claves: electroporacién, N. meningitidis, eficiencia de transformacién Introduction ‘Transformation is curently the most feasible option for exploring the genetics of Neisseria meningitidis, given that transducing phages, or efficient conjuga- tive systems, are not available for the Neisseriaceae [I]. Like other bacterial species, this organism isnatu- rally competent for genetic transformation. However, standard molecular techniques for transformation, used in Escherichia colt, are hardly applicable for 1N, meningitidis. The in vitro transformation of men- ingococeiis dependent on a numberof bacterial func- tions, most notably: the host pilition status, and the presence of species-specific sequence moti in the transforming DNA. Both factors affect the first stages of DNA transfer, during which nucleic acids traverse the bacterial envelope [2,3]. Unfortunately, even as few as two rounds of laboratory culture passages can render neiserial strains refractory to transformation, ‘due to the quick appearance and take over of non piliated, non competent variants (4). This situation is sometimes compounded by the absence of uptake ‘motif in the genetic constructions under study. Previously reported transformetion in Neisseria gonorrhoeae, using electroporation, support this tech- ‘nique as well suited for overcoming the above limita- tions [5]. Many species have been transformed by us- ing this methodology (6, 7]. Campylobacter jejuni [8] and E.coli(9] have served as models for field strength, time constant (pulse duration, temperature, host cells electroporation media, and DNA concentration opti- mization. However, despite the relatively simple con- cepts that apply to electroporation, the relatively com- 23 Corresponding author plicated or unknown parameters, inherent to cach bio- Togical sytem under study demand a clase monitor- ing of eleevoporation conditions. In this article we describe the development of a novel and simple clectoporation protocol to transform nonpliated var- ants ofthe N, meningidis stain B385. Materials and Methods Bacterial strains and culture conditions uded XL-1 Blue, nonpiliated vari- ant from N. meningitidis strain B385 (B4-P 1.15). cal cells were grown in a Brain Heart In- fusion (BHI) broth, or on BHI agar plates, at 37°C in ‘candle jar. XL-I Blue was grown in a Luria-Bertani (LB) broth. When needed, Ampicilin, or Kanamycin, ‘was added at a final concentration of 50 jg/mL. Recombinant DNA techniques ‘The methods used for in vitro manipulation of DNA. (plasmid purification, restriction analysis, Southern blots) were performed according to Sambrook, e¢ al [10}. Chromosomal DNA extraction from meningo- cocci was performed following the procedure de- seribed by Cornelissen et al, 1992 [11] Immunological methods Expanded colonies from the transformation plates, were inoculated intg $*mL of BHI cultures over- night at 37 °C. The following day, the cultures were 247 1, Facv ©, Fusanapger M, Mays TE Sacoenl econo or aed in reorlcompetone for warfarin tegen dries 2. Gite ‘pent ‘fowor ecu tre ditnd marian ‘Note ender) 1909398: 0812 3. Geodian 50, Seeece Face in fant ne specie raion of a 15. Genco Ch, Chan C3 AB, Kop 2m Ok Mone Sk elation and hr hoes ht dtecine te wae ‘Tien fom besser ond hoor ‘ener cers) Gan ceil 199 aires 6 Moniotaam ® Scie BH. High a ‘eaney tarforisan of accor ‘croton by sncroperoon, Hur Ke 2. Few KN, Hea Harder W, Yoon: fn Gent AB igh sc Aotoatrmain of be Yoo Homers paimomhe Cur Gant 142590810, Santiago Duets, eal harvested and 20 yg of total cellular proteins were used for SDS-PAGE [12] and Western blotting [13] with MAbII4, a monoclonal antibody elicited against the C-terminal region of the Lpda protein. ‘The immune complexes were visualized by using 3-amino-9 ethyl-carbazole, Plasmids pM-150: pUCI8-derived recombinant plasmids ob- tained from subcloning e 9 kb Kpal-Sall library frag- ment from N. meningitis, interrupted by a 1.2 Kb Kanamyein resistance marker from pUC&4K [14]. ‘pM-110: pUCI8-derived recombinant plasmid containing one copy ofthe 1.2 kb Kanamyetn rsise tance marker, from pUCAK, flanked by 207 and 253 bp from 5° and 3' regions ofthe lpda gene. 'pM-84: Recombinant plasmid containing one ine tact copy ofthe (oda gene, in pUCIB. Electroporation protocol 300 mL of the BHI broth were inoculated from a fresh plate of B385p- to a final Desa = 0.1 (In Photoelectric colorimeter, Model AE330, EOW, LTD) and grown at 37°C to exponential phase (Dasa = 0.8-0.9). Cells were harvested by con- trfugation (6000 rpm, 4°C, 5 mia), suspended in 150 mL of ice-cold electroporation buffer (EB: glye- erol 15%; MgCl, I mM; Sucrose 272 mM; HEPES, 1 mM; pH 7.2), and washed twice with 40 ant 20 mln the same EB. The cells were suspended in @ final volume of 400 yl. EB. High density aliquots (over 10° colony forming units (cfu/ml) of cell suspension (40 pL), were frozen at -70 °C or mixed with 1 uL of undigested plasmid DNA in TE 0.1X solution, and transfered immediatly 0.2 em electrode gap pre-chilled Gene Pulser eur vette (0.1 cm electrode gap cuvettes were used 10 reach field strengths above 12.5 kV/cm), just prior to the electric pulse with the BioRad Gene Pulser The parameters were set in the following ranges: Voltage, 8.0-14 kV/cm; Capacitance, 25-50 uP; Re- sistance, 100-600 Q. After pulsing, 1 mL of BHI was immediately added to the eels, followed by 3 h of incubation at 37 °C before plating and selec- {ion for Kanamycin resistance. The cells were plated both onto a selective me- dium forthe detection of plasmid recipients and onto anon selective medium for the determination of sure vivel rate. Transformants were visible 16-20 h after plating, and individual colonies were farther isolated by seriall streaking twice onto a selective medium before additonal analysis Results Determination of succeeding electroporation parameters The effect of different field strengths on the electroporation efficiency of B385p- was determined using |g of the plasmid pM-150, a single pulse ‘with a capacitance of 25 uF and a resistance of 400 resulting in pulse lengths of 6-9 ms. The best transformation efficiency was obtained using a single pulse of 10-12.5 kV/em. When the above conditions were used, strain B385p- consis- 248 Electroporation of Neisseria meningitidis tently gave 10° transformants/ug with a 60% sur- vival rate. Higher field strengths (13-15 kV/em) re- sulted in survival rates lower than 30% and thus a decrease in overall transformation efficiency. Lower values (8-9.5 kV/cm), while yielding higher survival rates (90%), were unable to achieve the same elec- twoporation frequency, even when longer pulse lengths were used (data not shown) Figure 1 shows the influence of pulse length in transformation efficiency, using the above conditions ‘and a constant field strength of 12.5 kV/cm. The best pulse time peaks were around 8.31 ms, with a drop in transformation efficiency above 8.78 ms. To ascertain the dependence of he electroporation cfficacy on the total amount of DNA, several assays ‘were set up using a single pulse of 11.5 kV/em with 1 capacitance of 25 uF and a resistance of 400 Q. The results for varying amounts of DNA are shown | sie Fn ° is pe fre) . gure 1. fect of ie pulse ia ansfomation eficency. Dato wereld aig th felowng endo cas ety xpenentolphaze grown ina gue BH medium) ware Sib pore ploumld DNA and cecroported wah fad Firongh of 12:5 fem In Figure 2. As can be seen, there is a steep increase in efficiency which levels off at $0 ng and starts to decrease above 200 ng. Pulse lengths of 6.3 to 7.7 ims were reached within the useful 50-200 ng range. ‘This further improvement allowed us to obtain more than 10° transformantsug of plasmid DNA, Generation of neisserial knock-out IpdA mutants ‘We applied these parameters to obtain neisserial/pdA knock-out mutants, A plasmid containing a Kanamy- DNA amet (i) Figure 2 Effect ofthe mount of DNA on transformation eff ciency Biotecnologia Apiads 1998; VOLIS, No. 4 Comte: ‘cert th lama OMA Poe el ‘ead $US 1980 95 856 430. 2, Dower. lle Rogie High sitcleny toclornaon of © cot Oy 1o.Sambros J Fitch EF Maniatis Stcond Eaton ela spring on ttcralry Pan, Clé Spring Horr New ade 1908 1.Gorneesen CH, Snes GO, ei arch OX Tomgron Sk, Spang 12.Laemm UE, Caoroge of sca rg the sey of oe hea [Foderioghge Ta Narre 1970.2 13. Town H Socal, Gordon le Sheut: procadre and some appears Proc Na Aco Sa USA TOPO 00" 14. iis 1 eating J The BUC pe tndh ant Impede fern ‘sith syhate nivorel pime's Gene Santiago Duefas, ea. cin resistance marker flanked by 207 and 253 bp from 5} and 3' regions of the ada gene (pM-110, ee Ma- terials and Methods) was used to electroporate 'B385p-. Since pM-I10 does not replicate in menin- gococei, Kanamycin resistant transformants can be rescued only in the event of a single (insertion and gene duplication) or double (allele replacement) re= combination between the sequenees flanking the se~ lesion mars and ihr geome countepar, Al eight transformants tested by Western blot with MAb14 (Figure 3) failed to express the protein, suge gesting that allelic replacement had ceeured in all cases. This conclusion was gontlrmed by Southern blot analysis of 4 transformants (Pigure 4) with an internal 300 bp Clal fragment from the (pd obtained from pM-84 a probe. Discussion We searched for an efficient electrotransformation protocol of N. meningitidis s a fessible way for the ‘generation of knock-out insertional mutants of ehror rmosomal genes. As @ model we used a 9 kb Kpnl= Sall library fragment from N. meningitidis B385 ine ‘erupted by a Kanamycin resisiange marker, With pM-150, the best results were obtained by using cells grown on aliquid BHI medium, harvested dure ing the early exponential phase, washed and suse pended jn the referred electroporation buffer, in aliquots of cell density above 10° efwlmL, which ‘were mixed with $0-200 ng of DNA in a low con- ductivity solution. A single electric pulse of 11.5 kV field strength reaching nearly 8 ms In length with a capacitance and resistance of 25 yl and 400 0, re- spectively, was applied. Field strength and pulse length were selected ac- cording to @ compromise between transformation ef ficiency and survival rate, Extreme values of these electrical parameters drastically reduced cell viabil- ity or failed to induce high DNA aquisition, respec- tively Figure 3. Wester blot of whole neisseril ells for LpdA pro- fein detection. Lane: 1, BIBS wid ype; 2-9, electroporated Colonies from BHI Kenomycin agar Plates. The double band present in the control is due to crosereaclvy of he common, Fpol binding domain present at las, in two dHfront daly

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