Archaea: a laboratory manual (original) (raw)

IVA cloning: A single-tube universal cloning system exploiting bacterial In Vivo Assembly

Scientific Reports, 2016

In vivo homologous recombination holds the potential for optimal molecular cloning, however, current strategies require specialised bacterial strains or laborious protocols. Here, we exploit a recAindependent recombination pathway, present in widespread laboratory E.coli strains, to develop IVA (In Vivo Assembly) cloning. This system eliminates the need for enzymatic assembly and reduces all molecular cloning procedures to a single-tube, single-step PCR, performed in <2 hours from setup to transformation. Unlike other methods, IVA is a complete system, and offers significant advantages over alternative methods for all cloning procedures (insertions, deletions, site-directed mutagenesis and sub-cloning). Significantly, IVA allows unprecedented simplification of complex cloning procedures: five simultaneous modifications of any kind, multi-fragment assembly and library construction are performed in approximately half the time of current protocols, still in a single-step fashion. This system is efficient, seamless and sequence-independent, and requires no special kits, enzymes or proprietary bacteria, which will allow its immediate adoption by the academic and industrial molecular biology community. Molecular cloning is at the heart of biomedical and biotechnological research, fundamental to protein structure-function studies, protein engineering and synthetic biology 1-3. Since the advent of the polymerase-chain reaction (PCR) 4 , cloning has involved selective PCR amplification and modification of DNA segments, which require directed assembly into a plasmid carrier for propagation in E. coli. Traditionally, restriction enzymes and ligases have been used to direct the assembly of DNA fragments 1 ; however, their sequence-dependency and laborious protocols led to the development of new alternatives, such as: PCR-only 5-8 , ligation-independent cloning (LIC) 9 , recombination-based 10,11 and multi-enzyme construction methods (such as Gibson 12 , InFusion 13 and USER 14). LIC, multi-enzyme construction and some recombination-based approaches (such as SLiCE 10) rely on in vitro enzymatic treatment of DNA fragments for assembly. While PCR-only methods eliminate the need for such enzymatic treatment, they involve multiple rounds of PCR and DNA purification. Both cases involve lengthy, hands-on protocols. In vivo assembly, where the bacterial host performs the fusion of DNA fragments, would eliminate the need for multiple steps and reagents, providing significant advantages over all current methods. The advantages of in vivo recombination have led molecular biologists to use the yeast gap-repair cloning system 15 , despite the hurdles associated with eukaryotic work. In vivo assembly in E. coli has previously been limited to the use of strains with enhanced recombinase activities, such as recA 16 and phage recombinases Red/ ET 17,18 , yet these bacterial strains enhance plasmid instability or require specialised preparation of competent cells. As a consequence, strains with reduced recombinase activities (e.g. recA knockouts) are universally used for molecular cloning. The presence of a recA-independent homologous recombination pathway in E. coli was reported more than 20 years ago 19-21 , but has been neglected until recently, except for sporadic use in specific high throughput applications 22,23. The pathway is mostly uncharacterised but is most efficient at recombining linear DNA fragments, likely acting through an annealing mechanism 20,24 , although alternative mechanisms have been suggested 25,26. Conveniently, the recA-independent pathway is responsible for the recombination of short overlapping sequences 19 , whereas the recA system requires longer homologous DNA stretches (> 150-300 bp). The pathway's short homology requirements, ubiquitous presence in laboratory E. coli strains (in our and others' experience 27-29) and reduced compromises on plasmid stability, make it an optimal tool for molecular cloning.

λ ZAP: a bacteriophage λ expression vector within vivoexcision properties

Nucleic Acids Research, 1988

A lambda Insertion type cDNA cloning vector, Lambda ZAP, has been constructed. In E. coBa phagemld, pBtuescript SK(-), contained within the vector, can be excised by 11 or M13 helper phage. The excision process elmlnates the need to subcbne DNA inserts from the lambda phage into a plasmld by restriction digestion and igation. This is possible because Lambda ZAP incorporates the signals for both initiation and termination of DNA synthesis from the 11 bacteriophage origin of repfication (1). Six of 21 restriction sites In the excised pBluescript SK polyBnker, contained within the NH2-portion of the lacZ gene, are unique in lambda ZAP. Coding sequences inserted into these restriction sites, in the appropriate reading frame, can be expressed from the lacZ promoter as fusion proteins. The features of this vector significantly increase the rate at which clones can be isolated and analyzed. The lambda ZAP vector was tested by the preparation of a chicken liver cDNA Hbrary and the isolation of actin clones by screening with oBgonucleotide probes. Putative actln clones were excised from the lambda vector and Identified by DNA sequencing. The ability of lambda ZAP to serve as a vector for the construction of cDNA expression libraries was determined by detecting fusion proteins from clones containing glucocerbrosidase cDNA's using rabbit IgQ anti-glucocerbrosidase antibodies.

High-throughput plasmid cDNA library screening

Nature Protocols, 2006

Libraries of cDNA clones are valuable resources for analysing the expression, structure, and regulation of genes, as well as for studying protein functions and interactions. Fulllength cDNA clones provide information about intron and exon structures, splice junctions and 5'-and 3'-untranslated regions (UTRs). Open reading frames (ORFs) derived from cDNA clones can be used to generate constructs allowing expression of native proteins and Nor C-terminally tagged proteins. Thus, obtaining full-length cDNA clones and sequences for most or all genes in an organism is critical for understanding genome functions. Expressed sequence tag (EST) sequencing samples cDNA libraries at random, which is most useful at the beginning of large-scale screening projects. However, as projects progress towards completion, the probability of identifying unique cDNAs via EST sequencing diminishes, resulting in poor recovery of rare transcripts. We describe an adapted, high-throughput protocol intended for recovery of specific, full-length clones from plasmid cDNA libraries in five days. 100X Purified BSA (10mg/mL) New England Biolabs (www.neb.com), Catalog #B9001S Adenosine 5'-triphosphate disodium salt Sigma (www.sigmaaldrich.com), Catalog #A-3377 Sequence-specific primers (20µM) Invitrogen/Illumina (www.invitrogen.com) T4 DNA Ligase (400U/µl) New England Biolabs (www.neb.com), Catalog #M0202L Dpn I (20U/µl) New England Biolabs (www.neb.com), Catalog #R0176L Deoxynucleotide Solution Mix New England Biolabs (www.neb.com), Catalog #N0447L Finnzymes Phusion™ High-Fidelity DNA Polymerase (2U/µl) New England Biolabs (www.neb.com), Catalog #F-530L 5X Phusion™ HF Buffer Supplied with Phusion™ polymerase, above TAM-1 Competent Cells Active Motif (www.activemotif.com), Catalog #11096 LB/Antibiotic Agar Plates LB Broth Base Invitrogen (www.invitrogen.com), Catalog #12780-029 Bacto Agar BD (www.bd.com), Catalog #214010 50mg/ml (stock concentration) antibiotic (Chloramphenicol [Roche (www.rocheapplied-science.com), Catalog #10634433001] is used for the pOT2 vector) 2× YT Media, dehydrated. VWR Scientific (www.vwrsp.com), Catalog #90003-330 BD Biosciences (www.bdbiosciences.com), Catalog #244020 Plasmid cDNA Library User supplied CRITICAL Successful screening depends on high quality, high complexity libraries; only clones that are present in a cDNA library may be recovered. Sephadex™ G-50, Superfine Amersham (www.amershambiosciences.com), Catalog #17-0041-01 BigDye™ Terminators v3.1 Cycle Sequencing Kit Applied Biosystems (www.appliedbiosystems.com), P/N 4337455 Sequencing Buffer, 5X Applied Biosystems (www.appliedbiosystems.com), P/N 4336697 Equipment 100mm x 15mm Petri Dish BD Falcon (www.bd.com), Catalog #351029 Applied Biosystems 9700 Thermal Cycler Applied Biosystems (www.appliedbiosystems.com), P/N 4314879 Innova 4300 Incubator Shaker New Brunswick Scientific (www.nbsc.com), Model #4300 MultiScreen-HV 96-well Plate, 0.45µm, hydrophilic, low protein binding, Durapore® membrane, clear, non-sterile with lid. Millipore (www.millipore.com), Catalog #MAHVN4550 MultiScreen Column Loader, 45µl, black, aluminum, non-sterile Millipore (www.millipore.com), Catalog #MACL09645 MultiScreen Centrifuge Alignment Frame, blue, aqueous applications Millipore (www.millipore.com), Catalog #MACF09604 MultiScreen Column Loader Scraper Millipore (www.millipore.com), Catalog #MACL0SC03 MicroAmp Base Applied Biosystems (www.appliedbiosystems.com), Catalog #N8010531 Soft-face hammer, 1.5 oz Stanley (www.stanleytools.com), Model #57-592 fitted with Tip #57-582. NOTE: any small, soft-face hammer will work.

219 - Lee 2011 J Biol Eng 5.12.pdf

Background: As engineered biological systems become more complex, it is increasingly common to express multiple operons from different plasmids and inducible expression systems within a single host cell. Optimizing such systems often requires screening combinations of origins of replication, expression systems, and antibiotic markers. This procedure is hampered by a lack of quantitative data on how these components behave when more than one origin of replication or expression system are used simultaneously. Additionally, this process can be time consuming as it often requires the creation of new vectors or cloning into existing but disparate vectors. Results: Here, we report the development and characterization of a library of expression vectors compatible with the BglBrick standard (BBF RFC 21). We have designed and constructed 96 BglBrick-compatible plasmids with a combination of replication origins, antibiotic resistance genes, and inducible promoters. These plasmids were characterized over a range of inducer concentrations, in the presence of non-cognate inducer molecules, and with several growth media, and their characteristics were documented in a standard format datasheet. A three plasmid system was used to investigate the impact of multiple origins of replication on plasmid copy number. Conclusions: The standardized collection of vectors presented here allows the user to rapidly construct and test the expression of genes with various combinations of promoter strength, inducible expression system, copy number, and antibiotic resistance. The quantitative datasheets created for these vectors will increase the predictability of gene expression, especially when multiple plasmids and inducers are utilized.

Phasmids as effective and simple tools for construction and analysis of gene libraries

Gene, 1989

Phasmid lpMYF131, a hybrid of phage Iz vectors and plasmid pUC19, was constructed. The phasmid and its derivatives were shown to be efficient vectors for construction and analysis of gene libraries in Escherikhiu coli cells. The 1pMYF131 DNA molecule contains all the genes and regions essential for phage lytic development. The plasmid cannot be packaged either in the monomeric or the oligomeric form due to its specific length. Elongation of the DNA molecule by ligation with fragments of foreign DNA can make it packageable and this is easily detected by plaque formation. Hence, the procedures used to construct genomic libraries can be simplified by selection of only recombinant DNA molecules just at the time and on the basis of their packaging in vitro. The output of recombinant clones per vector molecule was several times higher for vector ApMYF131, compared to phage vector 1L47.1AB, and attained 3 x lo6 clones per pg DNA. Vector and recombinant phasmids can be obtained in large quantities in plasmid form. lpMYF131 contains nine unique restriction sites which allow the cloning of DNA fragments with blunt ends and of fragments with various types of cohesive ends, obtained by digestion with 14 prototype restriction enzymes. The maximal size of the cloned DNA fragments is approx. 20 kb for llpMYF 13 1. Phasmid vectors were used to construct libraries of bovine, pig and quail genomes, and genomic libraries of 17 species of bacteria. Application of suitable methods allowed the identification 13 individual genes within these libraries.