Construction of Cry1Ac Plasmid Vector and Its Transformation Into Agrobacterium Tumefaciens

Introducing cry genesinto rice genome is reported able to produce rice plantresistant to stemborer. DNA sequence encodes cry1Ac genehas been inserted into pGEM4Z, but this construct does nothave a selectable marker gene for selection of transformedplant cells. The research aims were to construct a plasmidvector expressing a cry1Ac gene that has a transformationselectable gene and to transform it into Agrobacteriumtumefaciens. Materials used were pAY560325 binary plasmidvector, pGEM4Z-cry1Ac vector, Escherichia coli strain DH5-αand A. tumefaciens strain LBA4404 competent cells. Themethods consisted of plasmid DNA digestion using HindIIIand EcoRI, electrophoresis, DNA (backbone and insert)dissection from the gel, purification, and ligation using T4DNA ligase. Transformation of ligated DNA into E. coli byheat shock followed by cell plating onto selection medium,colony cultured, DNA isolation, and identification usingrestriction enzymes. Reconfirmation was done by cuttingusing restriction enzyme and PCR using F3 and R3, cry1Acgene specific primers. Research result were DNA fragmentsof 3.8 kb ubiquitin::cry1Ac insert and pAY560325, thebackbone vector, that after ligated and transformed into E.coli produced colonies. One of ten colonies containingplasmid DNA was evidently confirmed and namedpAY560325-cry1Ac. Subsequently, it was transformed into A.tumefaciens by electrophoration method. Plasmid DNA wasisolated from Agrobacterium that after digested with HindIIIand EcoRI produced DNA fragments of 9.44 kb (pAY560325)and 3.814 kb (ubiquitin::cry1Ac). While by PCR, plasmidproduced DNA fragment of about 711 bp. Thus, cry1Acplasmid vector (pAY560325-cry1Ac) was successfullyconstructed and transformed into A. tumefaciens and isready to be transformed into rice genome

Oxidative Stress Detection With Escherichia Coli Harboring A katG\u27::lux Fusion

A plasmid containing a transcriptional fusion of the Escherichia coli katG promoter to a truncated Vibrio fischeri lux operon (luxCDABE) was constructed. An E. coli strain bearing this plasmid (strain DPD2511) exhibited low basal levels of luminescence, which increased up to 1,000-fold in the presence of hydrogen peroxide, organic peroxides, redox-cycling agents (methyl viologen and menadione), a hydrogen peroxide-producing enzyme system (xanthine and xanthine oxidase), and cigarette smoke. An oxyR deletion abolished hydrogen peroxide-dependent induction, confirming that oxyR controlled katG\u27::lux luminescence. Light emission was also induced by ethanol by an unexplained mechanism. A marked synergistic response was observed when cells were exposed to both ethanol and hydrogen peroxide; the level of luminescence measured in the presence of both inducers was much higher than the sum of the level of luminescence observed with ethanol and the level of luminescence observed with hydrogen peroxide. It is suggested that this construction or similar constructions may be used as a tool for assaying oxidant and antioxidant properties of chemicals, as a biosensor for environmental monitoring and as a tool for studying cellular responses to oxidative hazards

T-DNA promoter tagging in Nicotiana tabacum : a thesis presented in fulfilment of the requirements for the degree of Master of Philosophy in Genetics at Massey University, Palmerston North, New Zealand

Plant development is primarily controlled at the level of gene expression. In order to analyse this regulation it is necessary to isolate genes which are involved in organ development through cellular and tissue determination or which respond to environmental signals. Promoter tagging was chosen in order to identify genes potentially associated with plant development by their spatial and temporal pattern of expression. The introduction of a promoterless reporter gene tag allows the expression patterns of plant genes to be readily characterised. A new series of promoter tagging vectors were constructed from the plasmid pPCV604 (Koncz, 1989). The selectable kanamycin resistance marker gene from pBin6 (Bevan, 1984) was cloned into pPCV604 to create pGT. The hygromycin phosphotransferase gene in pGT was then replaced with a promoterless (β-glucuronidase (gus) gene coupled with octopine synthase termination sequence subcloned from pKiwi101a (Janssen and Gardner, 1989) creating pGTG. This binary transformation vector required the helper pRK replication functions of Agrobacterium tumefaciens strain GV3101. In order to bypass this restriction, the vector sequence of pBin19 was combined with the T-DNA of pGTG to create pBin19-GTG. The latter plasmid was found to have a higher Agrobacterium tumefaciens-mediated Nicotiana tabacum transformation efficiency in strain LBA4404 than pGTG in strain GV3101. In both the pGTG and pBin19-GTG promoter tagging vectors the promoterless gus gene has an initiation codon 62 base pairs inside the T-DNA. This sequence includes translation termination codons in all three reading frames. Therefore, insertion of the T-DNA into a plant gene could lead to activation of the gus gene, under the control of the plant gene promoter, via transcriptional fusion. Nicotiana tabacum leaf segments were transformed with pGTG or pBin19-GTG and transgenic plants selected on kanamycin. A population of 87 transgenic tobacco plants were fluorometrically screened for GUS activity in leaf and root material; 37% were found to contain GUS activity, indicating a high frequency of promoter tagging. Two transgenic plants with root specific gus expression were analysed histochemically. Progeny after self-fertilisation lacked GUS activity, though this was restored in progeny of one plant with 5-azacytidine treatment, suggesting involvement of methylation in the gene silencing. Southern hybridisation, inverse PCR cloning of T-DNA flanking sequences and segregation on kanamycin indicated the presence of multiple T-DNA copies within the primary transformants. Furthermore, inverse PCR sequence from one plant indicated multiple and truncated T-DNA insertions at one or more loci. A further population of transformed plants was generated with pBin19-GTG and histochemically screened for GUS activity in roots (14 positive from 147 tested), shoots (27 positive from 147) and floral organs (14 positive from 56). Overall, combining results from all plant organs tested, an average of 33% of plants were found with GUS activity in one or more organs. A diverse range of patterns of gus expression were observed and described including patterns involving root branching. Forty four plants from this population were analysed for T-DNA copy number via Southern hybridisation with a gus probe (right border junction T-DNA) and nptII probe (central T-DNA). Multiple copies were frequently found with an average of 3.3 T-DNA copies per transgenic plant. Overall, an average of 11% of T-DNA insertions were found to be involved in gus activation. Comparison of the fluorometric (37% positive, 87 plants tested) and histochemical (22% positive, 147 plants tested) screens for GUS activity in root and shoot material was discussed and it is suggested that further care is needed in assigning promoter tagging hits from fluorometric screening. Variable expression was observed with promoter tagged genes. It is suggested that further research is required to determine whether this variation was due to silencing, perhaps by methylation, or was a result of the tagged promoters' normal expression patterns

Gateway vectors for efficient artificial gene assembly in vitro and expression in yeast Saccharomyces cerevisiae

Peer reviewedPublisher PD

Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains

Background: Homologous recombination mediated by the lambda-Red genes is a common method for making chromosomal modifications in Escherichia coli. Several protocols have been developed that differ in the mechanisms by which DNA, carrying regions homologous to the chromosome, are delivered into the cell. A common technique is to electroporate linear DNA fragments into cells. Alternatively, DNA fragments are generated in vivo by digestion of a donor plasmid with a nuclease that does not cleave the host genome. In both cases the lambda-Red gene products recombine homologous regions carried on the linear DNA fragments with the chromosome. We have successfully used both techniques to generate chromosomal mutations in E. coli K-12 strains. However, we have had limited success with these lambda-Red based recombination techniques in pathogenic E. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains. \ud \ud Results: Our goal was to develop a high-throughput recombineering system, primarily for the coupling of genes to epitope tags, which could also be used for deletion of genes in both pathogenic and K-12 E. coli strains. To that end we have designed a series of donor plasmids for use with the lambda-Red recombination system, which when cleaved in vivo by the I-SceI meganuclease generate a discrete linear DNA fragment, allowing for C-terminal tagging of chromosomal genes with a 6xHis, 3xFLAG, 4xProteinA or GFP tag or for the deletion of chromosomal regions. We have enhanced existing protocols and technologies by inclusion of a cassette conferring kanamycin resistance and, crucially, by including the sacB gene on the donor plasmid, so that all but true recombinants are counter-selected on kanamycin and sucrose containing media, thus eliminating the need for extensive screening. This method has the added advantage of limiting the exposure of cells to the potential damaging effects of the lambda-Red system, which can lead to unwanted secondary alterations to the chromosome. \ud \ud Conclusion: We have developed a counter-selective recombineering technique for epitope tagging or for deleting genes in E. coli. We have demonstrated the versatility of the technique by modifying the chromosome of the enterohaemorrhagic O157:H7 (EHEC), uropathogenic CFT073 (UPEC), enteroaggregative O42 (EAEC) and enterotoxigenic H10407 (ETEC) E. coli strains as well as in K-12 laboratory strains

Construction of a novel fungal gus expression plasmid, and its evaluation in Aspergillus nidulans : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University

A GUS expression plasmid, pFunGus, was constructed containing a multi-cloning site for the insertion of gene regulatory elements, to be used in fungal reporter gene studies. A derivative of pFunGus (pFG-gpd) was constructed by the insertion of the gpdA promoter (glyceradehyde-3-phosphatc dehydrogenase) into the multi-cloning site of pFunGus for the assessment of the plasmid's transformation and expression properties in Aspergillus niduans. The correct construction of pFunGus and pFG-gpd was verified by analytical restriction digests and by its property of GUS expression in A. nidulans. The plasmid was integrated into the A. nidulans genome via cotransformation with the phleomycin resistance plasmid, pAN8-l. Transformation frequencies of between 3 and 250 transformants per µg of pAN8-l DNA were obtained. Initial screening for cotransformation yielded no pFG-gpd transformants. Attempts to improve cotransformation frequencies by optimisation of cotransformation conditions were unsuccessful. However, large scale screenings of transformants lead to cotransformants being isolated at a very low cotransformation frequency. Approximately 0.45% of pAN8-l transformants possessed the GUS phenotype. The eight pFG-gpd transformants obtained were analysed by Southern hybridisation. Six out of the eight transformants had a single copy integration. Of the remaining two transformants, one had three copies integrated at separate locations, one of which was disrupted, and the other had four copies integrated as tandem repeats, one of which was disrupted. All the transforming DNA appeared to be integrated ectopically. The physiology of the transformants was assessed by dry weight increase, colony extension and total protein content. These showed that the transformants biology was not significantly compromised by the transforming DNA. Finally, high levels of GUS expression were observed in all pFG-gpd transformants and the GUS expression per copy of the GUS expression cassette integrated into the genome was constant. These results showed that the transformed gene copy number determined the levels of gene activity rather than the position of integration in the genome. Overall these results demonstrate the potential application of the versatile GUS expression plasmid, pFunGus for reporter gene studies in filamentous fungi

Membrane topology of the ArsB protein, the membrane subunit of an anion-translocating ATPase

The ars operon of the conjugative R-factor R773 encodes an oxyanion pump that catalyzes extrusion of arsenicals from cells of Escherichia coli. The oxyanion translocation ATPase is composed of two polypeptides, the catalytic ArsA protein and the intrinsic membrane protein, ArsB. The topology of regions of the ArsB protein in the inner membrane was determined using a variety of gene fusions. Random gene fusions with lacZ and phoA were generated using transposon mutagenesis. A series of gene fusions with blaM were constructed in vitro using a beta-lactamase fusion vector. To localize individual segments of the ArsB protein, a ternary fusion method was developed, where portions of the arsB gene were inserted in-frame between the coding regions for two heterologous proteins, in this case a portion of a newly identified arsD gene and the blaM sequence encoding the mature beta-lactamase. The location of a periplasmic loop was determined from V8 protease digestion of an ArsA-ArsB chimera. From analysis of data from 26 fusions, a topological model of the ArsB protein with 12 membrane-spanning regions is proposed

Novel particulate vaccine candidates recombinantly produced by pathogenic and nonpathogenic bacterial hosts : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology at Massey University, Manawatu, New Zealand.

Polyhydroxyalkanoates (PHAs) are biopolyesters synthesized as small spherical cytoplasmic inclusion bodies by a range of bacteria. Recently, PHA beads have been investigated for use as a vaccine delivery platform by using engineered heterologous production hosts that allowed the efficient display of vaccine candidate antigens on the beads surface and were found to greatly improve immunogenicity of the displayed antigens. However, like other subunit vaccines, these antigen-displaying (vaccine) PHA beads only provide a limited repertoire of antigens. In this thesis we investigate the idea of directly utilizing the disease causative pathogen or model organism to produce vaccine PHA beads with a large antigenic repertoire. These beads are hypothesized to have the potential to induce greater protective immunity compared to production of the same PHA bead in a heterologous production host. This concept was exemplified with Pseudomonas aeruginosa and Mycobacterium tuberculosis as model human pathogens. For P. aeruginosa we describe the engineering of this bacterium to promote PHA and Psl (polysaccharide) production. This represents a new mode of functional display for the engineering, production, and validation of a novel OprI/F-AlgE fusion antigen-displayed on PHA beads. For the disease tuberculosis we investigated the use of nonpathogenic M. smegmatis as a model organism for M. tuberculosis. We described the bioengineering, production, and validation of Ag85AESAT- 6 displayed on PHA beads produced in M. smegmatis. Here we showed that both organisms were harnessed to produce custom-made PHA beads for use as particulate subunit vaccines that carried copurifying pathogen-derived proteins as a large antigenic repertoire and the ability of these vaccine PHA beads to generate a protective immune response. This novel bioengineering concept of particulate subunit vaccine production could be applied to a range of pathogens naturally producing PHA inclusions for developing efficacious subunit vaccines for infectious diseases

Reprogramming Hansenula polymorpha for penicillin production: expression of the Penicillium chrysogenum pcl gene

We aim to introduce the penicillin biosynthetic pathway into the methylotrophic yeast Hansenula polymorpha. To allow simultaneous expression of the multiple genes of the penicillin biosynthetic pathway, additional markers were required. To this end, we constructed a novel host–vector system based on methionine auxotrophy and the H. polymorpha MET6 gene, which encodes a putative cystathionine β-lyase. With this new host–vector system, the Penicillium chrysogenum pcl gene, encoding peroxisomal phenylacetyl-CoA ligase (PCL), was expressed in H. polymorpha. PCL has a potential C-terminal peroxisomal targeting signal type 1 (PTS1). Our data demonstrate that a green fluorescent protein–PCL fusion protein has a dual location in the heterologous host in the cytosol and in peroxisomes. Mutation of the PTS1 of PCL (SKI-COOH) to SKL-COOH restored sorting of the fusion protein to peroxisomes only. Additionally, we demonstrate that peroxisomal PCL–SKL produced in H. polymorpha displays normal enzymatic activities.