Complete Automation of Lysis and Neutralization Steps Leads to High Quality Plasmid Midiprep Purifications

Transcription

1 Complete Automation of Lysis and Neutralization Steps Leads to High Quality Plasmid Midiprep Purifications ABSTRACT: The development of an automated plasmid midiprep method for use as a benchtop system has remained a challenge in the automation industry. The complex lysis and neutralization steps involved in almost every plasmid midiprep purification require skilled laboratory professionals to perform the sensitive procedures. In addition to the long time required to perform typical midiprep purifications, problems such as clogging, low yields, and carry over contamination can readily occur if care is not taken. Here, we describe a novel method for fully automated plasmid midiprep DNA purifications using the AutoPlasmid MEA system. The automated lysis and neutralization steps have been fully optimized, resulting in a sensitive, robust method yielding samples suitable for transformation, transfection, and sequencing. INTRODUCTION: Purification processes for plasmid DNA include many similar initial steps. After the transformed bacteria cells are harvested, an alkaline lysis procedure releases the plasmid DNA into solution. The original method for extraction of plasmid DNA from Escherichia coli was described by Birnboim and Doly (1). This method involves adding an alkaline buffer containing detergent to a resuspended cell pellet. Once the bacteria cell are released into solution, the highly viscous lysate is neutralized by addition of an acidic buffer containing a chaotropic salt. The formation of a white precipitated material is indicative of the neutralization reaction. The precipitate, or flocculate, consists mainly of cell debris, denatured proteins, and genomic DNA. While the long, unwound genomic DNA molecules remain intertwined within the flocculate, the circular plasmid DNA renatures quickly and remains in solution (1). A separation step is required in order to isolate the solution from the rest of the precipitated cell debris. Common techniques include centrifugation and filtration. For high throughput production of plasmid DNA, the use of these techniques involve long processing times, inconsistent performance, and a lack of scalability. This can result in inefficient purifications procedures capable of having a severe economic impact on the overall process. Although bench-top instruments for automated miniprep isolations of plasmid DNA have been around for some time, a system for processing midiprep culture volumes has yet to be developed. Due to the sensitive nature of the lysis, neutralization, and separation steps, an ideal automated midiprep method should satisfy three main criteria. Frist, the timing of the lysis and neutralization steps should be carefully controlled. Overexposure to the lysis and neutralization solutions can lead to irreversible plasmid degradation and the formation of unwanted isomers. Second, the buffers added during the process should be thoroughly mixed. A homogenous solution is crucial during each step to ensure maximum release of plasmid DNA from the cells, and to avoid local areas of ph-extremes (2). Third, the sample should be processed gently in order to avoid irreversible plasmid degradation and disintegration of the flocculate. Release of genomic DNA and other cell impurities from the flocculate into solution will lead to separation issues during the later chromatographic binding steps (3). A process that satisfies these criteria will result in a more pure final DNA product. In this report, thirteen 5 ml culture volumes containing plasmid DNA were purified using the AutoPlasmid MEA purification system from PhyNexus. The pre-programmed midiprep methods are capable of processing between 5 ml and 20 ml culture volumes, with minimal set up time, and consistent results between samples. The results displayed here show how an automated bench-top midiprep process can be an asset for any researcher performing routine plasmid purifications.

2 MATERIALS AND METHODS: Growth conditions Strain JMG001 was generated by transforming Escherichia coli DH5α competent cells with puc19, a high-copy plasmid of kb with Ampicillin resistance. Strain JMG002 was generated by transforming Escherichia coli DH5α competent cells with psg001, a medium-copy plasmid of with Ampicillin resistance. Frozen glycerol stocks of JMG001 and JMG002 were used to generate single colonies on 10 cm LB agar plates supplemented with ampicillin at 100 µg/ml. Two aliquots of 50 ml TB (Terrific broth) medium containing 100 µg/ml ampicillin (Teknova, A9501) were inoculated with single JMG001 and JMG002 colonies. The cultures were grown in a 250 ml Thomson Ultra Yield Flask (Thomson, ) with AirOTop Enhanced Seal (Thomson, ) at 40 C with shaking at 350 rpm in an incubated shaker (New Brunswick Scientific, G24 Environmental Incubator Shaker). Determination of cell density Cell densities (OD 600 ) were measured using a spectrophotometer (Milton Roy, Spectronic 601). Once the absorbance values reached a calculated OD value of greater than 10, 5 ml aliquots were removed from the cultures and prepared for plasmid DNA extraction. Cell culture aliquots were centrifuged at 3500 g (Dupont, Sorvall T6000B) for 30 min, and the supernatant liquid was decanted entirely. Sample preparation For each 5 ml sample of pelleted cells, 200 µl of resuspension buffer was used. Resuspension was performed manually using wide orifice pipette tips. Samples were mixed until no visible cell clumps remained. Eight of the resuspended samples containing the high-copy plasmid were transferred to row A on a 96-deep well plate. The other five resuspended samples containing the medium-copy plasmid were transferred to row A on a separate 96-deep well plate. Purification on the AutoPlasmid MEA system For all thirteen samples, the AutoPlasmid MEA system deck was prepared according to the Midiprep 1 Row (1-12 samples) pre-programmed method. Before running the method, the 96-deep well plates containing the resuspended pellets were placed in position 3. An empty 96-deep well plate was placed in position 5 to collect the eluted plasmids. A reservoir plate was placed in position 7, and an 8x1 plate was placed in position 8. Buffers were added to the specified plate positions according to the deck layout (figure 1B) Pipette tips were placed in the tip box in position 2. The total set up time after resuspension took approximately 5 minutes. After the deck was prepared, the method was executed through the computer software. The cells containing the high-copy plasmid were processed first, and the purified plasmids were stored in sterilized tubes. After, the cells containing the medium-copy plasmid were processed, and both the purified plasmids and the capture flow-through were stored in sterilized tubes Analyzing capture flow-through The capture flow-through was analyzed by performing an additional purification on new PhyTip Columns. Instead of beginning from the lysis step, the method was started from the capture step. Spectroscopic analysis

3 A) The concentration of the purified plasmid DNA was determined using a Nanodrop spectrophotometer (ND-1000), and Quibit flourometer (ThermoFisher, Q33216). Purity of the samples was checked by the ratio of absorbance at 260:230 nm and 260:280 nm as measured by the Nanodrop. Double-stranded DNA concentration was verified using the using a Quibit Broad range assay (ThermoFisher, Q10210). Agarose gel electrophoresis An agarose gel electrophoresis analysis (90V, 60 min) was performed using 1.0% agarose gels in in TAE (40 mm Tris base, 20 mm acetic acid, and 1 mm EDTA, ph = 8.0) buffer. The gels were stained in 0.5 µg/ml ethidium bromide for 15 minutes, and destained in deionized water for 15 minutes. Position 2 Position 3 Position 5 Position 7 Position 8 B) Figure 1: A) Top-down view of AutoPlasmid MEA deck with plates. B) Deck layout for Midiprep 1 Row (1-12 samples) method. The AutoPlasmid MEA deck was manually set up according the Midiprep 1 Row (1-12 samples) Autoplasmid MEA method. Wash Buffer (100 ml) was added to the reservoir plate in position 7. Lysis buffer (10 ml), Precipitation Buffer 1 (10 ml), Precipitation Buffer 2 (5 ml), deionized water (15 ml), and Elution Buffer (15 ml), were added to rows 1-5 of the 8x1 plate in position 8. Pipette tips were placed in the tip box in positon 2. PhyTip Columns were placed in row 1, LT1000 tips were placed in rows 2-4, and wide orifice tips were place in row 8.

4 RESULTS: Purification of high-copy plasmids on the AutoPlasmid MEA system Cultures were inoculated with single bacterial colonies transformed with DH5α-pUC19 plasmid. Cultures were grown for approximately 16 hours with shaking at 40ºC. The cultures were processed as described and analyzed by UV spectroscopy and agarose gel electrophoresis (Figure 2). Yields were consistent, averaging 68.5 μg recovered. DNA was visualized on an agarose gel and purity measured by A 260 /A 280 and A 260 /A 230 ratios, which were in acceptable ranges, between , respectively. Yields and quality were in line with performance measured using competitive technology with the same starting culture volume. Lane # Conc. (ng/µl) Yield (µg) A260/ A260/ A280 A Figure 2: Analysis of plasmid DNA purified with Lysate Direct PhyTip Columns. DNA quality was qualitatively assessed by agarose gel analysis of non-linearized medium-copy plasmid (DH5α-pUC19) purified using Lysate Direct PhyTip Columns (Left). Purified samples were run on a 1% agarose gel stained with ethidium bromide (right). DNA quality was quantitatively assessed spectroscopically using a nanodrop device and quibit assay (left). Sample eluent was produced using 800 µl elution buffer. Lane 9 contains purified plasmid using a midiprep kit from Supplier Q. Reaching full equilibrium through back and forth capture Before the capture step takes place, the robotic pipette head transfers the neutralized solution to a new well. From here, the Lysate Direct PhyTip Columns are used. A combination of hydrophobic interactions, hydrogen bonding, and electrostatic shielding allow for the binding of plasmid DNA onto the silica resin (4). Unlike spin column or filter based formats that only have one quick binding step, the method used with PhyTip Columns employs back and forth flow. This allows for the solution to be passed through the resin multiple times in order to reach full binding equilibrium. Figure 3 displays how increasing the number of capture cycles correlates with higher yields, and a smaller amount of plasmid in the flow through. By employing a method with 16 back and forth capture cycles, all of the plasmid DNA will be bound to the PhyTip Column.

5 Lane Capture Conc. Yield A260/ A260/ Sample Cycles (ng/µl) (µg) A280 A Eluent Capture flow-through Eluent Capture flow-through Eluent Capture flow-through Eluent Capture flow-through Eluent Capture flow-through Figure 3: Analysis of plasmid DNA purified with Lysate Direct PhyTip Columns. DNA quality was qualitatively assessed by agarose gel analysis of nonlinearized medium-copy plasmid (DH5α-Psg001) purified using Lysate Direct PhyTip Columns (Left). Purified samples were run on a 1% agarose gel stained with ethidium bromide. DNA quality was quantitatively assessed spectroscopically using a nanodrop device and quibit assay (Right). Sample eluent was produced using 800 µl elution buffer. Capture flow through samples were processed on a second PhyTip Column, and eluted in 150 µl of buffer. DISCUSSION: Many researchers involved in routine plasmid purifications fail to realize the impact that slight procedural variations may have on the quality and quantity of plasmid DNA during lysis and neutralization steps. Despite a researcher s careful attention to detail, the nature of manual lysis and naturalization steps using tubes and containers allow for more human error. In order to produce optimal results for midiprep plasmid purifications, automated systems are ideal for maintaining reproducibility, allowing for scalability, and increasing efficiency. The methods developed for midiprep plasmid purifications on the AutoPlasmid MEA system have been carefully designed to maximize quality, yield, and time efficiency. A successful lysis procedure will limit plasmid degradation, cell disintegration, and genomic DNA breakage, while releasing the full contents of the bacteria cells (5). On the AutoPlasmid MEA, the lysis step is completed within 3 minutes, keeping the contact time consistent between samples. The flow rate through the wide orifice pipette tip is precisely controlled, simultaneously releasing the full contents of the bacteria cells while eliminating potential areas of ph-extremes. The volumes that are aspirated and expelled, and the flow rate through the wide orifice pipette tip have been optimized to ensure gentle and homogenous mixing of the entire solution. Through careful control of these variables, neither incomplete lysis nor over lysis occurs. During neutralization, the topology of the plasmid DNA in solution plays an important role. After the cells have been lysed, an acidic solution is added to the lysate. While the supercoiled plasmid DNA renatures quickly, the long genomic DNA molecules remain intertwined with denatured proteins, detergent, and other cellular macromolecules. These unwanted molecules form a complex precipitate, or flocculate, that is visible within the solution. Chaotropic substances are commonly used to help lyse cells and inactivate enzymes, and create a favorable binding environment for nucleic acids onto silica. Similar to the lysis step, inadequate or rough mixing can lead to increased amount of contaminants in the plasmid solution. These contaminants have been known to interfere with the later chromatographic separations (3). Once the mixture has been fully neutralized, the majority of contaminating molecules will be present in the precipitated material. A separation step is needed in order to separate the plasmid DNA still in

6 solution. Centrifugation and filtration are the most frequently used methods. Despite their widespread use, centrifugation is difficult to scale, and filtration often results in low yields or clogged membranes. Careful timing is required for a successful separation. Homogenous yet gentle mixing is needed in order for full precipitation of the flocculate to occur without causing its degradation. The methods developed for Midiprep plasmid purifications on the AutoPlasmid MEA have also been designed for complete automation of the neutralization step, without the use of a centrifuge or filtration device. Precise buffer design, effective flow rate control, and rigorous experimental testing has led to a system capable of outperforming most manual procedures. The entire step occurs within an 8 minute time frame, leading to faster processing compared to any centrifugation or filtration technique. Through the innovative technology of the PhyTip Column, solutions can be passed through a resin bed as many times as needed. This allows for complete equilibrium binding of plasmid DNA to silica resin contained inside the columns. By utilizing a controlled capture step of 16 back and forth cycles, no plasmid DNA remains in the flow through. After utilizing a similar process for the washing steps, the sample is eluted off the column and collected in a deep well plate. CONCLUSION: Midi prep scale plasmids are prepared directly from the cell pellet with a bench top instrument. The AutoPlasmid MEA benchtop instrument may purify up to 36 samples per run, processing the samples 12-at-a-time. REFERENCES: (1) Birnboim, H. C., and Doly, J A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl. Acids Res. 7: (2) Liou, J. T., Shieh, B. H., Chen, S.W., and Li, C An improved alkaline lysis method for minipreparation of plasmid DNA. Prep. Biochem. Biotechnol. 29: (3) M.M. Diogo, J.A. Queiroz, D.M.F. Prazeres. Chromatography of plasmid DNA. J. Chromatography A. 2005, 1069 (2005) 3-22 (4) Melzak, K. A.; Sherwood, C. S.; Turner, R. F. B.; Haynes, C. A. Driving forces for DNA adsorption to silica in perchlorate solutions. J. Colloid Interface Sci. 1996, 181 (2), (5) Kieser, T Factors affecting the isolation of CCC DNA from Streptomyces lividans and Escherichia coli. Plasmid. 12:19 36.