DOCSLIB.ORG

Synthesis of a Contiguous 32-Kb Polyketide Synthase Gene Cluster

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Synthesis of a Contiguous 32-Kb Polyketide Synthase Gene Cluster Total synthesis of long DNA sequences: Synthesis of a contiguous 32-kb polyketide synthase gene cluster Sarah J. Kodumal, Kedar G. Patel, Ralph Reid, Hugo G. Menzella, Mark Welch, and Daniel V. Santi* Kosan Biosciences, Inc., 3832 Bay Center Place, Hayward, CA 94545 Communicated by Robert M. Stroud, University of California, San Francisco, CA, September 17, 2004 (received for review July 19, 2004) To exploit the huge potential of whole-genome sequence infor- Our efforts to this end stemmed from a desire to develop mation, the ability to efficiently synthesize long, accurate DNA heterologous expression of large polyketide synthase (PKS) sequences is becoming increasingly important. An approach pro- genes in Escherichia coli. Type I modular PKS genes encode the posed toward this end involves the synthesis of Ϸ5-kb segments of giant enzymes (among the largest proteins known) that synthe- DNA, followed by their assembly into longer sequences by con- size polyketide natural products such as erythromycin, epothi- ventional cloning methods [Smith, H. O., Hutchinson, C. A., III, lone, and tacrolimus (12). These genes reside within the high Pfannkoch, C. & Venter, J. C. (2003) Proc. Natl. Acad. Sci. USA 100, GϩC genomes of the actinomycete and myxobacterial groups of 15440–15445]. The major current impediment to the success of this prokaryotes and encode proteins with multiple sets, or modules, tactic is the difficulty of building the Ϸ5-kb components accurately, of active sites (domains). Each module catalyzes the assembly of efficiently, and rapidly from short synthetic oligonucleotide build- a specific two-carbon-unit component of the polyketide product. ing blocks. We have developed and implemented a strategy for the We sought to recreate PKS genes with the twin objectives of high-throughput synthesis of long, accurate DNA sequences. Un- optimizing their codon composition for efficient expression in E. purified 40-base synthetic oligonucleotides are built into 500- to coli and to introduce common restriction sites flanking modules 800-bp ‘‘synthons’’ with low error frequency by automated PCR- and domains that would permit facile interchangeability, thus based gene synthesis. By parallel processing, these synthons are exploiting the full potential of combinatorial biosynthesis of efficiently joined into multisynthon Ϸ5-kb segments by using only ‘‘unnatural natural products’’ (12). three endonucleases and ‘‘ligation by selection.’’ These large Smith et al. (4) proposed building very long DNA sequences by segments can be subsequently assembled into very long sequences synthesis of Ϸ5-kb segments of DNA from short synthetic oligo- by conventional cloning. We validated the approach by building a nucleotides, followed by their assembly into longer sequences by synthetic 31,656-bp polyketide synthase gene cluster whose func- conventional methods. However, methodologies for preparing seg- tionality was demonstrated by its ability to produce the megaen- ments of Ϸ5 kb were not sufficiently accurate or facile to enable zyme and its polyketide product in Escherichia coli. implementation of the approach. For the large number of se- quences to be synthesized in our project, we therefore excluded the he chemical synthesis of genes and genomes has received possibility of one-step synthesis of the Ϸ5-kb segments, because this Tconsiderable attention for several decades and is becoming would have required time-consuming, manual correction of nu- increasingly important in the exploitation of whole-genome se- merous errors (4). Instead, we developed methods to build them in quence information. The field was pioneered by Khorana and two error-free steps. First, we constructed multiple perfect se- coworkers with the then-heroic total synthesis of tRNA structural quences Ϸ500 bp in length called ‘‘synthons’’† (13); then we used a genes (1, 2) and by Itakura et al. (3) with the synthesis and facile method, dubbed ligation by selection (LBS), to connect them expression of the somatostatin gene. Since then, DNA synthesis into multisynthon segments of Ϸ5,000 bp. These segments, in turn, methodology has made steady progress, with current approaches were readily assembled into larger sequences by conventional relying on the enzyme-catalyzed assembly of short, chemically cloning strategies, as illustrated by our construction of a contiguous, synthesized oligonucleotides. Of the various methods, polymerase synthetic 31.7-kb PKS gene cluster. cycling assembly (PCA) (4) is the most widely used because of its inherent simplicity. Overlapping, complementary oligonucleotides Materials and Methods are annealed and recursively elongated with a heat-stable DNA Enzymes were obtained from New England Biolabs, unless other- polymerase to ultimately yield a full-length sequence, which is wise noted, and used as recommended. Molecular biological tech- amplified by conventional PCR. PCA, first reported for synthesis of niques were used as standard protocols (14). The pUC18-derived the 303-bp HIV-2 Rev gene (5), has since evolved (6–8) into a plasmids pKOS239-172-2 and pKOS293-172-A76 were reported in widely used general method for synthesis of genes of up to Ϸ1 kb. ref. 15. DH5␣ E. coli was made chemically competent with a kit BIOCHEMISTRY The 1-kb size barrier was broken in 1990 by Mandecki et al. (9), from Zymogen Research (Orange, CA). Oligonucleotides were who synthesized a 2.1-kb plasmid by ligation of 30 fragments, and from Qiagen͞Operon Technologies (Alameda, CA). NTPs were again in 1995 when Stemmer et al. (7) reported the one-step PCA PCR-grade from Roche Applied Sciences. DNA sequencing was synthesis of a 2.7-kb plasmid that was purified by antibiotic performed on an ABI 3730 DNA analyzer (Applied Biosystems) selection. Smith et al. (4) assembled the 5,386 ␾X174 bacterio- according to the manufacturer’s recommended protocol. phage genome from a single pool of chemically synthesized oligonucleotides by using a combination of ligation and PCA methods, but purification of the product again required biolog- Abbreviations: DEBS, 6-deoxyerythronolide B synthase; EF, error frequency; LBS, ligation by ical selection. In 2002, Cello et al. (10) described a stepwise selection; LIC, ligation-independent cloning; PCA, polymerase cycling assembly; PKS, polyketide synthase; TU, transcription unit; UDG͞LIC, uracil DNA glycosidase͞ligation- synthesis of a 7,558-bp poliovirus cDNA by ligation and PCA. independent cloning. This sequence appears to be the longest synthetic DNA reported Data deposition: The sequences in this paper have been deposited in the GenBank database to date. Visionaries have even projected application of DNA (accession nos. AY661566 and AY771999). synthesis technology to build synthetic, minimal genomes (11). If *To whom correspondence should be addressed. E-mail: [email protected]. such goals are to be realized, methods will be needed to prepare †In synthetic chemistry, synthons are defined as ‘‘structural units within a molecule which long, contiguous, and perfect sequences of DNA without re- are related to possible synthetic operations’’ (13). quiring biological selection for purification. © 2004 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0406911101 PNAS ͉ November 2, 2004 ͉ vol. 101 ͉ no. 44 ͉ 15573–15578 Downloaded by guest on October 1, 2021 For uracil DNA glycosidase͞ligation-independent cloning to provide cohesive 4-nt 5Ј overhangs for ligation. Oligonucleotides (UDG͞LIC), the forward primer was 5Ј-GCUAUAUCGCUAUC- of 40 bases were synthesized that collectively encoded both strands GAUGAGCUGCCACTGAGCACCAACTACG, and the reverse of the insert, each having 20-nt overlaps with 40-mer oligos from the primer was 5Ј-GCUAGUGAUCGAUGCAUUGAGCUG- opposite strand. The single-stranded 5Ј overhangs were allowed to GCACTTCGCTCACTACACC. vary in size and filled during assembly. Gene synthesis and sequencing were assisted by an inte- grated automation system consisting of a BioMek FX, a robotic Synthon Synthesis. Oligonucleotide consolidation and assembly. To each ORCA arm, and a tip lift (Beckman Coulter), plate sealer and well of a microtiter plate was added 5 ␮lofa50␮M solution (250 piercer (Velocity11), Palo Alto, CA), two tetrad thermal pmol) of each of the oligonucleotide components of a synthon, and cyclers (MJ Research, Cambridge, MA), and a cytomat hotel sufficient water was added to double the volume. For synthons up (Kendro, Asheville, NC). to 1 kb, each well of the ‘‘assembly’’ microtiter plate was loaded with 48 ␮l of a stock solution containing 0.5 ␮l of Expand High Fidelity Vector Construction. The BsaI site in the apramycin-resistance polymerase (5 units͞␮l, Roche), 1.0 ␮l of 10 mM dNTPs, 5.0 ␮lof R (Ap ) genes of pKOS293-172-2 and pKOS293-172-A76 were 10ϫ PCR buffer, 3.0 ␮l of 25 mM MgCl2, and 38.5 ␮l of water. To changed to GAGATC by PCR-based SDM (16) to give separate wells of the assembly plate, 2.0 ␮l of each oligonucleotide pKOS309-52 [ApR and chloramphenicol-resistance (CmR)] and mixture was added. For synthons Ͼ1 kb, additional oligonucleotide pKOS309-53 [ApR and kanamycin-resistance (KmR)], respectively. mixture was added to keep the final concentration of individual The tetracycline-resistance gene (TetR), obtained from pACYC184 oligonucleotides at 1 ␮M. Thermal cycling began with a 5-min by PCR, was introduced into the EcoRV site of pKOS309-52 with denaturing step at 95°C, and continued with 25 cycles at 95°C for the 5Ј end of the gene adjacent to the ApR gene to generate 30 s, 50°C for 30 s, and 72°C for 90 s. pKOS399-16-78 (ApR,CmR, and TetR). By using PCR-based Amplification. Each well of the ‘‘amplification’’ microtiter plate was site-directed mutagenesis, the BbsI site in the TetR gene of loaded with 48.75 ␮l of a stock solution containing 0.5 ␮l of Expand pKOS399-16-78 was changed to GTATTC to give pKOS399-21-1, High Fidelity polymerase (5 units͞␮l, Roche), 1.0 ␮l10mM R and the EcoRI site in the Cm gene was changed to GAGTTC to dNTPs, 5.0 ␮l10ϫ PCR buffer, 3.0 ␮l of 25 mM MgCl2, 39.25 ␮l give pKOS399-51-1.
Load more

Recommended publications
  • Improving DNA Plasmid Production in Escherichia Coli
    Improving DNA Plasmid Production in Escherichia Coli by ADAM SINGER (Under the Direction of Mark A. Eiteman) ABSTRACT The ability to produce large quantities of plasmid DNA is imperative for wide scale availability of DNA vaccines. Large scale, high yield production relies on the synergy between host strain, plasmid, medium and production scheme. Screening as many variables as quickly and cost effectively as possible is the goal. In this study, Escherichia coli strains were transformed with two plasmids and screened for plasmid yield in shake flasks in chemically defined medium supplemented with either glucose or glycerol. High yield candidates were grown in feed batch fermentations at two specific growth rates, µ = 0.14 h-1 and µ = 0.24 h-1. As predicted, high production in shake flasks was predictive of high production in fermentations. Using our media and process, we were able to reach volumetric yields of approximately 600 mg/L and specific yields of approximately 17.82 mg/g, regardless of growth rate. We were also able to increase productivity (mg/Lh) over 30%. INDEX WORDS: E. coli, fed-batch, gene therapy, plasmid production Improving DNA Plasmid Production in Escherichia Coli by ADAM SINGER B.S., Biological Engineering, University of Georgia, 1998 A Thesis Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE ATHENS, GEORGIA 2007 © 2007 Adam Singer All Rights Reserved Improving DNA Plasmid Production in Escherichia Coli by ADAM SINGER Major Professor: Mark A. Eiteman Committee: Elliot Altman Sidney Kushner Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia August 2007 iv DEDICATION To my wife Dana and my daughter Sydney-Rose.
    [Show full text]
  • A Visual Demonstration of Plasmid DNA Preparation from Bacteria
    HOW-TO-'DO-IT A Visual Demonstration of Plasmid DNA Preparation from Bacteria * RALPH L. KEIL, LAURA K. PALMER Downloaded from http://online.ucpress.edu/abt/article-pdf/71/6/363/55200/20565332.pdf by guest on 02 October 2021 Unprecedented advances in unraveling mysteries of life have disease, or other areas the students or instructor want to develop. occurred as a result of the molecular biology revolution. Some Since many students know someone with diabetes (in many cases major achievements of this revolution include new methods for the they know a classmate who has diabetes or one of them may even production of therapeutic compounds, insights into mechanisms suffer from the disease), this example provides an opportunity to of gene function and regulation, and the complete sequencing of get students to actively participate in discussion, regardless of their the genomes of numerous organisms including humans. A critical academic abilities. foundation of this revolution is the ability to use the bacterium An alternative introduction is to discuss consequences of Escherichia coli (E. coli) as a factory to produce large amounts of sequencing the entire three billion base pairs of DNA of the virtually any DNA of interest by inserting it into a small, extrachro human genome (http://www.ornl.gov/sci/techresources/Human mosomal circle of DNA called a plasmid. The ease and reproducibil Genome/home.shtml). The current and potential uses of this ity of plasmid DNA isolation from bacteria permits numerous labs technology in forensic science, health care, and business decisions to conduct molecular biology experiments and greatly accelerates are all areas that may be discussed depending on what focus the progress in understanding complex biological processes.
    [Show full text]
  • Control of Ph During Plasmid Preparation by Alkaline Lysis of Escherichia Coli
    Analytical Biochemistry 378 (2008) 224–225 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio Notes & Tips Control of pH during plasmid preparation by alkaline lysis of Escherichia coli Cheri Cloninger, Marilyn Felton 1, Bonnie Paul 1, Yasuko Hirakawa, Stan Metzenberg * Department of Biology, California State University, Northridge, Northridge, CA 91330, USA article info abstract Article history: Alkaline lysis of Escherichia coli is usually the method of choice for plasmid preparation, but ‘‘ghost bands” Received 9 March 2008 of denatured supercoiled DNA can result if the pH is too high or the period of lysis is too long. By replacing Available online 11 April 2008 the usual sodium hydroxide lysis solution with an arginine buffer prepared in the range of pH 11.4 to 12.0, we were able to stabilize the pH during lysis and obtain plasmid that is suitably pure for restriction digestion and DNA sequencing. Ó 2008 Elsevier Inc. All rights reserved. The extraction of plasmids from Escherichia coli is one of the most 2. To each 1 ml of cell suspension, 1 ml of a lysis solution consist- commonly used methods in molecular biology, and detergent lysis of ing of 1% (w/v) sodium dodecyl sulfate (SDS) and 0.5 M L-argi- cells in 0.1 M sodium hydroxide (NaOH,2 final) is the most wide- nine (pH 11.7) is added, and the tube is capped and rocked spread approach [1,2]. The alkalinity denatures the chromosomal briefly to mix. The lysate is allowed to sit undisturbed for 5 min.
    [Show full text]
  • Molecular Biology Services Price List Denmark; Valid from 01.09.2017
    Molecular Biology Services Price List Denmark; valid from 01.09.2017 Gene Synthesis Service Delivery times for Gene Synthesis Service: Standard Genes up to 1000 bp: 6 business days; guaranteed: 12 business days Standard Genes ≤ 3000 bp: 15 - 20 business days Standard Genes > 3000 bp: 4 additional business days/kb; Subcloning: additional 5 - 10 business days Express Genes up to 1000 bp: 4 business days Express Genes up to 1500 bp: 6 business days; up to 2000 bp: 7 days; Express Genes up to 3000 bp: 11 business days; up to 4000 bp: 13 days; Express subcloning: 4 business days Delivery time for complex gene varies. Dependent on the complexity of the gene, usually it takes 5-10 business days longer than estimated for standard genes. Part ID Service Description Price [DKK] 5001-000010 Short Standard Genes (≤ 500 bp) 1,125.00 / gene 5001-000016 Standard Genes (501-1000 bp) 2.40 / base pair 5001-000012 Long Standard Genes (1001-3000bp) 2.40 / base pair 5001-000019 Long Standard Genes (3001-4000bp) 2.70 / base pair 5001-000119 Long Standard Gene (4001-5000bp) 3.00 / base pair 5001-000219 Long Standard Gene (5001-6000bp) 3.15 / base pair 5001-000319 Long Standard Gene (6001-7000bp) 3.53 / base pair 5001-000419 Long Standard Gene (7001-8000bp) 3.53 / base pair 5001-000519 Long Standard Gene (8001-9000bp) 3.53 / base pair 5001-000619 Long Standard Gene (9001-10000bp) 3.53 / base pair 5001-000719 Long Standard Gene > 10 kb on request 5001-000021 Complex Genes on request 5001-000230 Express Fee Short Standard Gene (≤ 500 bp) 3,000.00 / gene 5001-000232
    [Show full text]
  • Genomic/Plasmid Dna and Rna)
    NPTEL – Bio Technology – Genetic Engineering & Applications MODULE 4- LECTURE 1 ISOLATION AND PURIFICATION OF NUCLEIC ACIDS (GENOMIC/PLASMID DNA AND RNA) 4-1.1. Introduction Every gene manipulation procedure requires genetic material like DNA and RNA. Nucleic acids occur naturally in association with proteins and lipoprotein organelles. The dissociation of a nucleoprotein into nucleic acid and protein moieties and their subsequent separation, are the essential steps in the isolation of all species of nucleic acids. Isolation of nucleic acids is followed by quantitation of nucleic acids generally done by either spectrophotometric or by using fluorescent dyes to determine the average concentrations and purity of DNA or RNA present in a mixture. Isolating the genetic material (DNA) from cells (bacterial, viral, plant or animal) involves three basic steps- • Rupturing of cell membrane to release the cellular components and DNA • Separation of the nucleic acids from other cellular components • Purification of nucleic acids 4-1.2. Isolation and Purification of Genomic DNA Genomic DNA is found in the nucleus of all living cells with the structure of double- stranded DNA remaining unchanged (helical ribbon). The isolation of genomic DNA differs in animals and plant cells. DNA isolation from plant cells is difficult due to the presence of cell wall, as compared to animal cells. The amount and purity of extracted DNA depends on the nature of the cell. The method of isolation of genomic DNA from a bacterium comprises following steps (Figure 4-1.2.)- 1. Bacterial culture growth and harvest. 2. Cell wall rupture and cell extract preparation. Joint initiative of IITs and IISc – Funded by MHRD Page 1 of 57 NPTEL – Bio Technology – Genetic Engineering & Applications 3.
    [Show full text]
  • An Improved System for Competent Cell Preparation and High Efficiency Plasmid Transformation Using Different Escherichia Coli Strains
    Electronic Journal of Biotechnology ISSN: 0717-3458 Vol.8 No.1, Issue of April 15, 2005 © 2005 by Pontificia Universidad Católica de Valparaíso -- Chile Received June 8, 2004 / Accepted January 13, 2005 TECHNICAL NOTE An improved system for competent cell preparation and high efficiency plasmid transformation using different Escherichia coli strains Zhiming Tu China-UK HUST-Res Crop Genetics Engineering and Genomics Joint Laboratory School of Life Science and Technology Huazhong University of Science and Technology Wuhan, 4300074, China Tel: 86 27 87556214 Fax: 86 27 87548885 E-mail: [email protected] Guangyuan He* China-UK HUST-RRes Crop Genetics Engineering and Genomics Joint Laboratory School of Life Science and Technology Huazhong University of Science and Technology Wuhan, 4300074, China Tel: 86 27 87556214 Fax: 85 27 87548885 E-mail: [email protected] Kexiu X. Li China-UK HUST-RRes Crop Genetics Engineering and Genomics Joint Laboratory School of Life Science and Technology Huazhong University of Science and Technology Wuhan, 4300074, China Tel: 86 27 87556214 Fax: 86 27 87548885 Mingjie J. Chen China-UK HUST-RRes Crop Genetics Engineering and Genomics Joint Laboratory School of Life Science and Technology Huazhong University of Science and Technology Wuhan, 4300074, China Tel: 86 27 87556214 Fax: 86 27 87548885 Junli Chang China-UK HUST-RRes Crop Genetics Engineering and Genomics Joint Laboratory School of Life Science and Technology Huazhong University of Science and Technology Wuhan, 4300074, China Tel: 86 27 87556214 Fax: 86 2787548885 Ling Chen China-UK HUST-RRes Crop Genetics Engineering and Genomics Joint Laboratory School of Life Science and Technology Huazhong University of Science and Technology Wuhan, 4300074, China Tel: 86 27 87556214 Fax: 86 2787548885 Qing Yao China-UK HUST-RRes Crop Genetics Engineering and Genomics Joint Laboratory School of Life Science and Technology Huazhong University of Science and Technology Wuhan, 4300074, China Tel: 86 27 87556214 Fax: 86 2787548885 Dongping P.
    [Show full text]
  • SGI-DNA-Bioxp-Lucigen-Webinar
    Accelerate your Research with Synthetic DNA and Hands-free Cloning Brought to you in collaboration with Lucigen and SGI-DNA Introduction Julie K. Robinson Synthetic Genomics, SGI-DNA Sr. Product Manager, Synthetic Biology The Potential of Synthetic Biology C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 2 Synthetic Genomics Collaborative Efforts Advance C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 3 Advancing Genomic Research Solutions, SGI-DNA Reagents Instruments ● Gibson Assembly® enables rapid assembly ● BioXp™ 3200 – an automated, benchtop of multiple DNA fragments genomics workstation ● Vmax electrocompetent cells for protein ● Create genes, genetic elements and complex expression genetic sequences ● Uses electronically transmitted sequence to build genes Services Bioinformatics ● Synthesize custom DNA of varying size ● Expert bioinformatics support, which and complexity includes whole genome annotation, gene expression analysis, and other types of ● Utilizes patented Gibson Assembly® sequence design method and error correction technology ● Archetype® software enables ‘omics-based ● Cell engineering services understanding of data to enable researchers ● NGS and plasmid preparation services to discover, analyze and build genes C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C. | 4 Common Genomic applications Applications of DNA Cloning Study of Genomes & Transgenic Gene Expression Gene Therapy Organisms Biopharmaceutical Recombinant Protein Cell Engineering Research Production C O P Y R I G H T © 2 0 1 7 S Y N T H E T I C G E N O M I C S , I N C.
    [Show full text]
  • Bacterial Culture & Plasmid Purification
    Genetic Tools and Reagents Bacterial Culture Media, Agar, Yeast Extract, Casein Peptone Plasmid Purification Bacterial Culture & Plasmid Purification Product Manual Bacterial Culture Media Protocols & Principle Plasmid Purification Protocols & Principle Bacterial Culture Media & Omni-Pure™ Mini-Prep Plasmid DNA Purification System For research use only. Not for use in diagnostic procedures for clinical purposes. Ordering Information Bacterial Culture Media Product Catalog No. Unit Size Agar Type A Bacterial Culture Grade, 100 g 40-3301-10 100 g Agar Type A Bacterial Culture Grade, 500 g 40-3301-05 100 g Agar Type A Bacterial Culture Grade, 1 kg 40-3301-01 1 kg Yeast Extract Bacterial Culture Grade, 100 g 40-4331-10 100 g Yeast Extract Bacterial Culture Grade, 500 g 40-4331-10 500 g Yeast Extract Bacterial Culture Grade, 1 k g 40-4331-10 1 kg Casein Peptone (Type 1) Bacterial Culture Grade, 100 g 40-3305-10 100 g Casein Peptone (Type 1) Bacterial Culture Grade, 500 g 40-3305-05 500 g Casein Peptone (Type 1) Bacterial Culture Grade, 1 kg 40-3305-01 1 kg Omni-Pure Plasmid DNA Purification Systems; Mini-Prep Product Catalog No. Unit Size* Omni-Pure Plasmid DNA Purification System; Mini-Prep 40-4020-01 100 Omni-Pure Plasmid DNA Purification System; Mini-Prep 40-4020-05 500 *Mini-prep plasmid purification. M_Bacterial_Culture Page 2 of 20 Bacterial Culture Media & Omni-Pure™ Mini-Prep Plasmid DNA Purification System For research use only. Not for use in diagnostic procedures for clinical purposes. Bacterial Culture & Plasmid Purification Protocols
    [Show full text]
  • Bacterial Transformation
    TECHNIQUES IN MOLECULAR BIOLOGY – BACTERIAL TRANSFORMATION The uptake of exogenous DNA by cells that alters the phenotype or genetic trait of a cell is called transformation. For cells to uptake exogenous DNA they must first be made permeable so the DNA can enter the cells. This state is referred to as competency. In nature, some bacteria become competent due to environmental stresses. We can purposely cause cells to become competent by treatment with chloride salts of metal cations such as calcium, rubidium or magnesium and cold treatment. These changes affect the structure and permeability of the cell wall and membrane so that DNA can pass through. However, this renders the cells very fragile and they must be treated carefully while in this state. Competent cells vary in how well they take up DNA. We can express this The amount of cells transformed per 1 µg of DNA is called the transformation efficiency. Too little DNA can result in low transformation efficiencies, but too much DNA also inhibits the transformation process. Transformation efficiencies generally range from 1 x 104 to 1 x 107 transformed cells per µg of added DNA. E. coli bacteria are normally poisoned by the antibiotic ampicillin. Ampicillin inhibits synthesis of the bacterial cell wall (in bacteria like E. coli , found between the inner and outer cell membranes), resulting in bacteria that are very structurally weak. In the hypotonic media in which these cells grow, cells exposed to ampicillin will swell and burst or not grow at all. For cells to survive, they must include a means to break down the ampicillin.
    [Show full text]
  • QIAGEN® Plasmid Purification Handbook
    February 2021 QIAGEN® Plasmid Purification Handbook QIAGEN Plasmid Mini, Midi, Maxi, Mega, and Giga Kits For purification of ultrapure, transfection-grade plasmid DNA Sample to Insight__ Contents Kit Contents ............................................................................................................... 3 Shipping and Storage ................................................................................................. 4 Intended Use .............................................................................................................. 4 Safety Information ....................................................................................................... 5 Quality Control ........................................................................................................... 5 Introduction ................................................................................................................ 6 Principle and procedure .................................................................................... 6 Equipment and Reagents to Be Supplied by User ............................................................ 8 Important Notes .......................................................................................................... 9 Protocol: Plasmid or Cosmid DNA Purification Using QIAGEN Plasmid Mini Kit .............. 17 Protocol: Plasmid or Cosmid DNA Purification Using QIAGEN Plasmid Midi and Maxi Kits .............................................................................................................................
    [Show full text]
  • Molecular Cloning
    Now includes Recombinant Albumin Buffers Molecular Cloning TECHNICAL GUIDE be INSPIRED drive DISCOVERY stay GENUINE OVERVIEW TABLE OF CONTENTS 3 Online Tools 4–5 Cloning Workflow Comparison 6 DNA Assembly Molecular Cloning Overview 6 Overview Molecular cloning refers to the process by which recombinant DNA molecules are 6 Product Selection produced and transformed into a host organism, where they are replicated. A molecular 7 Golden Gate Assembly Kits cloning reaction is usually comprised of two components: 7 Optimization Tips 8 Technical Tips for Optimizing 1. The DNA fragment of interest to be replicated. Golden Gate Assembly Reactions 2. A vector/plasmid backbone that contains all the components for replication in the host. 9 NEBuilder® HiFi DNA Assembly 10 Protocol/Optimization Tips ® DNA of interest, such as a gene, regulatory element(s), operon, etc., is prepared for cloning 10 Gibson Assembly by either excising it out of the source DNA using restriction enzymes, copying it using 11 Cloning & Mutagenesis PCR, or assembling it from individual oligonucleotides. At the same time, a plasmid vector 11 NEB PCR Cloning Kit is prepared in a linear form using restriction enzymes (REs) or Polymerase Chain Reaction 12 Q5® Site-Directed Mutagenesis Kit (PCR). The plasmid is a small, circular piece of DNA that is replicated within the host and 12 Protocols/Optimization Tips exists separately from the host’s chromosomal or genomic DNA. By physically joining the 13–24 DNA Preparation DNA of interest to the plasmid vector through phosphodiester bonds, the DNA of interest 13 Nucleic Acid Purification becomes part of the new recombinant plasmid and is replicated by the host.
    [Show full text]
  • Zyppy™ Plasmid Miniprep Kit
    INSTRUCTION MANUAL Zyppy™ Plasmid Miniprep Kit Catalog Nos. D4036, D4019, D4020 & D4037 (Patent Pending) Highlights Pellet-Free™ procedure* omits conventional cell-pelleting and resuspension steps. The fastest, simplest procedure for purifying the highest quality endotoxin-free plasmid DNA. Innovative colored buffers* permit error-free visual identification of complete bacterial cell lysis and neutralization. Contents Product Contents ................................................. 1 Product Description .............................................. 2 Product Specifications .......................................... 3 Buffer Preparation ................................................ 3 Protocol ................................................................ 4 Troubleshooting Guide ..................................... 5, 6 Ordering Information ............................................ 7 List of Related Products .................................. 8-10 * Patent pending; For Research Use Only Ver. 1.2.6 ZYMO RESEARCH CORP. Phone: (949) 679-1190 ▪ Toll Free: (888) 882-9682 ▪ Fax: (949) 266-9452 ▪ [email protected] ▪ www.zymoresearch.com Page 1 Product Contents: Note: Satisfaction of all Zyppy™ D4036 D4019 D4020 D4037 Storage Zymo Research products is Plasmid Miniprep Kit 50 preps. 100 preps. 400 preps. 800 preps. Temperature guaranteed. If you should be 7X Lysis Buffer*1 dissatisfied with this product 6 ml 12 ml 48 ml 2 x 48 ml Room Temp. please call 1-888-882-9682. (Blue) Neutralization 20 ml 40 ml 160 ml 2 x 160 ml 4-8 ºC Buffer*2 (Yellow) Endo-Wash Buffer 15 ml 30 ml 120 ml 2 x 120 ml Room Temp. Zyppy™ Wash Buffer 6 ml 12 ml 48 ml 2 x 48 ml Room Temp. (concentrate)3 Zyppy™ Elution 5 ml 5 ml 20 ml 2 x 20 ml Room Temp. Buffer Zymo-Spin™ IlN 50 2 x 50 8 x 50 16 x 50 Room Temp. Columns Collection Tubes 50 2 x 50 2x 200 4 x 200 Room Temp.
    [Show full text]