P UC

We created a pair of vectors allowing simple and efficient molecular cloning of any gene of interest with minimal consumption of time, labor and material. This system is applicable for standard molecular cloning, high-throughput cloning and generation of fusion protein libraries as well as for more complex gene assembly purposes. Also, this zero-background procedure allows going from cDNA to gene expression analysis in a defined vector in \2 days.

The South Pacific Journal of Natural and Applied Sciences, 2012

Current cloning technologies based on site-specific recombination are efficient, simple to use, and flexible. With the recent availability of complete genomic sequences of many organisms and plants, high-throughput and cost-efficient systems for gene cloning and functional analysis are in great demand. This study compares two different methods of preparation of competent cells using two strains of E.coli DH5α and HB101. From results the most efficient strain was found DH5α for cloning as it supports blue white screening utilizing galactosidase activity. The concentration of calcium chloride is another important factor; various concentrations of CaCl 2 were tried. Optimum concentration was found to be 75 mM. However PEG also has great influence on transformation efficiency, use of 40% PEG gave the best transformation efficiency. A convenient and rapid method for the genetic transformation of E.coli with appropriate plasmid is proposed which can be utilised for high efficiency transformation in normal laboratory conditions.

The main distinction between a living and non-living entity is the ability to replicate and reproduce similar offsprings. Nucleic acid molecules (DNA and RNA) present in a living organism acts as a genetic template to pass the hereditary information from one generation to the next. Nucleic acid molecules are organised as genes which code for a particular phenotype via specific proteins and the expression of a gene is regulated by both external and internal factors which aid the developmental process of an organism.

Lipase gene was aimed to be cloned from Bacillus subtilis 168 into the E.coli DH5α host. The strain of Baciilus subtilis 168 is ordered from the ATCC . primer designing was done by obtaining a sequence of the lipase gene from NCBI website, A fragment of lipase gene 212 bp in size was obtained using a pair of highly degenerate primers (estA FP and estA RP). the primers are designed to amplify the lipase gene from Bacillus subtilis 168 The used primers have the restriction sites on both ends for restriction enzymes ( BamHI and EcoRI). The targeted gene (lipase) was carried on a plasmid (PUC18) . PUC 18 plasmid was isolated from E.coli DH5 Alpha which has a poly linker sequence located within the lacZα provides several (10 in case of pUC18/pUC19) unique restriction sites for DNA insertion. The unique restriction sites used for integration of lipase gene insertion into pUC18 vectors interrupt the lacZα fragment so that appropriate E. coli cells possessing recombinant pUC DNA are β-galactosidase deficient and, as a result, produce white colonies on X-gal medium. The PCR product was successfully amplified by using optimizes PCR cycle. Competent cell preparation was done by using calcium chloride method, Positive transformants shows white colonies with associated antibiotic agar after overnight incubation at 37 ?C. The positive clones were streaked onto X-gal medium agar to screen for true lipase producers and were incubated overnight at 37 ?C. The recombinant clones formed an intense white color on the X-gal medium agar plate. The aim of the project is to ensure a successful transformation and expression of lipase gene that is used various applications ex: detergent industry and medical purposes etc. as the E. coli generation time has a faster growth rate and more stable than Bacillus spp.

Nucleic Acids Research, 1996

The genomic region encoding the type IIS restrictionmodification (R-M) system HphI (enzymes recognizing the asymmetric sequence 5′-GGTGA-3′/5′-TCACC-3′) from Haemophilus parahaemolyticus were cloned into Escherichia coli and sequenced. Sequence analysis of the R-M HphI system revealed three adjacent genes aligned in the same orientation: a cytosine 5 methyltransferase (gene hphIMC), an adenine N6 methyltransferase (hphIMA) and the HphI restriction endonuclease (gene hphIR). Either methyltransferase is capable of protecting plasmid DNA in vivo against the action of the cognate restriction endonuclease. hphIMA methylation renders plasmid DNA resistant to R.HindIII at overlapping sites, suggesting that the adenine methyltransferase modifies the 3′-terminal A residue on the GGTGA strand. Strong homology was found between the N-terminal part of the m6A methyltransferasease and an unidentified reading frame interrupted by an incomplete galE gene of Neisseria meningitidis. The HphI R-M genes are flanked by a copy of a 56 bp direct nucleotide repeat on each side. Similar sequences have also been identified in the non-coding regions of H.influenzae Rd DNA. Possible involvement of the repeat sequences in the mobility of the HphI R-M system is discussed.

Applied and Environmental Microbiology, 1994

Shuttle vectors (pMS3 and pMS4) which replicated in Escherichia coli and in gram-positive Acetobacterium woodii were constructed by ligating the replication origin of plasmid pAM,V1 with the E. coli cloning vector pUC19 and the tetM gene of streptococcal transposon Tn916. Electrotransformation of A. woodii was achieved at frequencies of 4.5 x 103 transformants per ,ug of plasmid DNA. For conjugal plasmid transfer, the mobilizable shuttle vector pKV12 was constructed by cloning the tetM gene into pAT187. Mating of E. coli containing pKV12 with A. woodii resulted in transfer frequencies of 3 x 10-6 to 7 x 10-6 per donor or recipient.

Bioluminescence as a light source, 2017

There are many things to consider in an experiment such as this one. The first step is to decide on the type of the bacteria that has the desired qualities in order to do this experiment. I have chosen to work with Escherichia coli (E. coli) for the main reasons that it is easier to obtain compared to other bacteria and that the growth rate and the generation time of E. coli is moderately fast, making it suitable for having results in a short time interval. In addition E. coli is grouped in under Biosafety Level 1 (BSL1), which makes it suitable for work involving well-characterized agents not known to cause disease in healthy adult humans, and of minimal potential hazard to laboratory personnel and the environment. A BSL1 lab doesn’t require any special design features beyond those suitable for a well-designed and functional laboratory. The strain used in this experiment is JM109 non-pathogenic, grows well and is transformed efficiently, it is useful for blue/white screening and it is also useful for cloning. The genotype of JM109 strain.

Nucleic Acids Research, 2003

The prophage of coliphage N15 is not integrated into the chromosome but exists as a linear plasmid molecule with covalently closed hairpin ends (telomeres). Upon infection the injected phage DNA circularizes via its cohesive ends. Then, a phageencoded enzyme, protelomerase, cuts the circle and forms the hairpin telomeres. N15 protelomerase acts as a telomere-resolving enzyme during prophage DNA replication. We characterized the N15 replicon and found that replication of circular N15 miniplasmids requires only the repA gene, which encodes a multidomain protein homologous to replication proteins of bacterial plasmids replicated by a theta-mechanism. Replication of a linear N15 miniplasmid also requires the protelomerase gene and telomere regions. N15 prophage replication is initiated at an internal ori site located within repA and proceeds bidirectionally. Electron microscopy data suggest that after duplication of the left telomere, protelomerase cuts this site generating Y-shaped molecules. Full replication of the molecule and subsequent resolution of the right telomere then results in two linear plasmid molecules. N15 prophage replication thus appears to follow a mechanism that is distinct from that employed by eukaryotic replicons with this type of telomere and suggests the possibility of evolutionarily independent appearances of prokaryotic and eukaryotic replicons with covalently closed telomeres.