Journal of Experimental Microbiology and Immunology (JEMI) Copyright © April 2015, M&I UBC Construction of an enlarged pUC19 vector with a rop gene designed to study plasmid maintenance in Escherichia coli Benson Chang, Arnab Ray, Thomas Tsuei, Rachel Wan Department of Microbiology and Immunology, University of British Columbia Experimental evidence has demonstrated that when pUC19 and pBR322 are co-transformed into DH5α Escherichia coli, only pUC19 is maintained after a few generations. Possible selective factors include the difference in size between the two plasmids, the presence of a negative regulator rop gene encoded by pBR322 and a G→A point mutation in the origin of replication of pUC19. The focus of this study was the construction of a novel enlarged pUC19 vector, pBART, which carries the rop and tetracycline resistance genes derived from pBR322. Previous attempts at producing a rop+ pUC19 were unsuccessful and failures were attributed to low DNA insert concentration and poor ligation. To circumnavigate these problems, the pBR322 fragment was amplified using PCR and through the process of primer design, a unique XbaI restriction site was created. The PCR fragment was digested and ligated into pUC19 and subsequently transformed into DH5α cells. Disruption of lacZ by inserting the pBR322 fragment into the multiple cloning site of pUC19 assisted in isolating transformants carrying the pBR322 fragment. The pBART construct was confirmed by restriction mapping and sequencing. The pUC19 derived pBART construct is comparable in size to pBR322 and can be used to explore how the role of plasmid size, presence of rop and the origin of replication G→A point mutation contributes to differences in plasmid copy number during cotransformation of pUC19 and pBR322. Plasmids pUC19 and pBR322 are common cloning deletion mutants of pBR322 to correspond closely in size vectors derived from the plasmid pCOlE1 (1). It has been to pUC19 (5, 6, 7). Other studies have attempted to explore experimentally observed that co-transformation of pBR322 the relevance of the rop gene by deleting or mutating the and pUC19 results in the selective maintenance of pUC19 gene from pBR322, but have been inconclusive due to over pBR322 in Escherichia coli DH5α cells (2). This unsuccessful ligation and low DNA insert concentrations observation has been attributed to a point mutation (GA) (8, 9,10, 11). Our study took a different approach as we in the origin of replication and lack of the rop gene in attempted to construct pBART, an enlarged pUC19 vector, pUC19 (1). In both pUC19 and pBR322, replication is by inserting the rop gene and tetracycline resistance gene regulated by the RNA II synthesis primer which anneals to from pBR322 into the multiple cloning site of pUC19 its complementary sequence at the origin of replication and (Figure 1).The insertion of the pBR322 fragment, provides a free 3’OH group for DNA extension after containing the rop and tetracycline resistance genes, RNase H cleavage (1). This process is down-regulated by increased the size of pUC19 from 2686 bp to 4989 bp, RNA I, which hybridizes to RNA II, and the Rop protein which is comparable to the 4361 bp size of pBR322, while further down-regulates DNA replication by stabilizing the still maintaining the GA point mutation in the original hybridization between both RNA I and II (1). The GA pUC19 origin of replication. Construction of the pBR322 point mutation in the origin of replication decreases the fragment required manipulation of restriction sites due to ability for RNA I to hybridize to RNA II, leading to a findings in previous studies which showed that use of the higher copy number of pUC19 than pBR322. Additionally, NdeI restriction site in pBR322 does not result in the size difference between pBR322 and pUC19 has been successful ligation (5). Instead, we created a unique XbaI suggested to have an impact on the difference in site in our pBR322 fragment for successful ligation into replication efficiency between the two plasmids since the pUC19 multiple cloning site. We hypothesize that maintenance and replication of multiple plasmids leads to plasmid size difference and presence of the rop gene metabolic burden on host cells (3). Previous decreases maintenance of pBR322 in DH5α cells and the transformation studies suggest that transformation construction of pBART will be effective in determining efficiency is inversely related to plasmid size as it has been the influence of these factors on the selective maintenance shown that pUC19 has a higher transformation efficiency of pUC19 over pBR322 when co-transformed. than pBR322 (4, 5). One explanation for this observation is Correspondingly, future studies using pBART will enable that the larger pBR322 plasmid requires more energy and better understanding of the influence of plasmid size and time to replicate than the pUC19 plasmid, consequently the presence of the rop gene on plasmid copy number resulting with lower plasmid copy numbers over time (2). differences between pUC19 and pBR322. Alternatively, preferential maintenance of pUC19 may result due to the metabolic inefficiency for host cells to MATERIALS AND METHODS maintain different plasmids containing the same antibiotic Bacterial strains and growth conditions. E. coli DH5α cells resistance gene against ampicillin (1). were obtained from the Ramey strain collection of the Previously, attempts to understand the effect of size on Department of Microbiology and Immunology at the University transformation efficiency have been made by creating of British Columbia and were grown in Luria-Bertani (LB) broth Page 1 of 5 Journal of Experimental Microbiology and Immunology (JEMI) Copyright © April 2015, M&I UBC or LB agar plates at 37oC. When appropriate, 100µg/ml ampicillin to allow for ligation of the two plasmid fragments. Restriction (Sigma-Aldrich) and 12µg/ml tetracycline (Sigma-Aldrich) was digest reactions were carried out using HindIII-HF®(New also added to the growth media for selection of DH5α cells England Biolabs) and XbaI (New England Biolabs) in containing plasmids pUC19 and pBR322, respectively. For CutSmartTM buffer (New England Biolabs) at 37oC for 2 hours. colony screening purposes, 2% w/v X-gal solution (Thermo Restriction enzymes were then heat inactivated at 65oC for 15 Scientific) and 20% w/v IPTG (isopropyl β-D- minutes. thiogalactopyranoside; Thermo Scientific) was also added to Agarose gel electrophoresis of pUC19 and pBR322 growth media when appropriate. fragments. The pUC19 and pBR322 fragments used to create the Isolation of plasmids. Overnight cultures of E. coli DH5α- pBART construct were visualized on an agarose gel to confirm containing pUC19 and pBR322, provided by the culture the identity of both fragments. The plasmid fragments were collection of the Department of Microbiology and Immunology at mixed with 6X DNA Loading Dye (Thermo Scientific) and the University of British Columbia, were grown at 37oC in LB loaded onto a 0.8% w/v agarose gel alongside a GeneRulerTM broth containing 100µg/ml ampicillin on a platform shaking at DNA Ladder Mix (Thermo Scientific) for band size comparison 180rpm. Isolation of plasmids pUC19 and pBR322 were and was run for 50 minutes at 100V in 1X TAE buffer. The gel performed using the Invitrogen PureLink® Quick Plasmid was stained in 0.5µg/ml ethidium bromide solution for 30 minutes Miniprep Kit (Life Technologies) following the manufacturer’s before visualization and imaging with Alpha Imager protocol. The plasmid DNA was eluted with sterile distilled water (MultimageTM Light Cabinet). and concentration was quantified using a Nanodrop 2000c Ligation of pUC19 and pBR322 fragments. Following the Spectrophotometer (Thermo Scientific). creation of sticky ends from the restriction digest of pUC19 and Preparation of competent E. coli DH5α cells. For the pBR322 fragments, the ligation of both fragments together was preparation of competent E. coli DH5α cells, SOB media was performed with Invitrogen T4 DNA Ligase (Life Technologies), prepared with 2% w/v tryptone, 0.5% w/v yeast extract, 8.56mM following the manufacturer’s protocol with the addition of a final NaCl, 2.5mM KCl and 10mM MgSO4 in distilled water and concentration of 5% w/v polyethylene glycol (PEG) 6000 to the sterilized via autoclaving. CCMB80 transformation buffer was ligation reaction. Ligation reactions were carried out at room o prepared with 10mM potassium acetate, 80mM CaCl2, 20mM temperature for 1 hour before being heat inactivated at 65 C for MnCl2, 10mM MgCl2, 10% v/v glycerol in distilled water, pH 15 minutes. The ligated products were then transformed via heat adjusted to 6.4 and filter sterilized via a 0.45µm pore size filter. shock into competent E. coli DH5α cells and spread onto LB agar E. coli DH5α cells were inoculated into 250ml SOB media and plates containing ampicillin, X-gal, and IPTG (LB-amp-Xgal- o grown to an OD600 of 0.3 at 37 C. The culture was then IPTG). centrifuged down at 4oC in order to pellet the cells. After Restriction digest map of putative pBART plasmid decanting the supernatant, the cell pellet was then resuspended in constructs. Putative pBART plasmid constructs were subjected 80ml CCMB80 transformation buffer, incubated on ice for 20 to a series of restriction enzyme digests in order to map out and minutes and then centrifuged down to pellet the cells again. confirm the identity of the plasmid construct. Isolated white Finally, after decanting the supernatant, the pelleted cells were colonies from the LB-amp-Xgal-IPTG plates were grown in then resuspended in 10ml CCMB80 transformation buffer and overnight cultures of LB broth containing ampicillin and plasmids aliquoted into 500µl vials before storage at -80oC until heat shock were isolated from each white colony using Invitrogen transformation. PureLink® Quick Plasmid Miniprep Kit (Life Technologies), Primer preparation and PCR conditions. Primers were following manufacturer’s protocol. The plasmids were eluted obtained from Integrated DNA Technologies®, Inc.