Genome shuffling methods were explored for Bacillus subtilis strain molecular breeding. Recycling protoplast fusion, recycling transformation and recycling universal transduction were used for genome shuffling in B. subtilis. Four strains with different nutrition-deficiency markers were used as initial strains. After five rounds protoplast fusion, transformation or transduction, the descendant with 4 markers had not been detected, and the rate of descendant with 3 markers were 4.53 x 10(-4), 1.64 x 10(-4), 4.47 x 10(-3), respectively. A computer program was made to simulate the recycling fusion process. Based on simulation result and comparing the genome shuffling result of B. subtilis in this experiment and that of Streptomyces coelicolor reported in references, effective genome shuffling needs a high recombination rate of at least between 10(-3) and 10(-2).
Bacillus subtilis ; classification ; genetics ; DNA Shuffling ; Genetic Techniques ; Genome, Bacterial ; genetics ; Protein Engineering ; Transformation, Bacterial

To develop a genetic transformation method of Aureobasidium pullulans and T-DNA insertion for high-efficient screening of polymalic acid (PMA) producing strain. Agrobacterium tumefaciens-AGL1, containing the selection genes encoding hygromycin B phosphotase or phosphinothricin acetyltranferase, was used to transform Aureobasidium pullulans CCTCC M2012223 and transformants were confirmed by colony PCR method. Transferred DNA (T-DNA) insertional mutants were cultured in microwell plate, and screened for high-titer PMA producing strain according to the pH response model. DNA walking was used to detect the insertion sites in the mutant. Results show that the selection markers could stably generated in the transformants, and 80 to 120 transformants could be found per 10(7) single cells. A high-titer PMA mutant H27 was obtained, giving a good PMA production caused by the disruption of phosphoglycerate mutase, that increased by 24.5% compared with the control. Agrobacterium tumefaciens-mediated transformation and high-efficient screening method were successfully developed, which will be helpful for genetic transformation of Aureobasidium pullulans and its functional genes discovery.
Agrobacterium tumefaciens ; Ascomycota ; genetics ; metabolism ; DNA, Bacterial ; Malates ; metabolism ; Polymerase Chain Reaction ; Polymers ; metabolism ; Transformation, Genetic

In order to optimize the conditions of construction BAC library, the transformation efficiency of E. coli DH10B was studied in this paper. Our data prove much higher competence of electroporation (reaches 2.19 x 10(10) cfu/microg pUC19 DNA) when harvesting the cells between an OD550 of 0.7 - 0.8. Five different electric field strength (from 9 kV/cm to 25 kV/cm) and three different sized plasmid vector DNAs including pUC19 DNA, pECBAC1 DNA and pCLD04541 DNA, as well as three bacterial artificial chromosomes (BACs) ranging from 40 to 190 kb and their mixture were used to discover the transformation efficiency changes under various conditions. Our data show maximum transformation efficiency and optimal electric field strength of plasmid DNAs drop dramatically with increasing size of the DNA. Molecules of 190 kb transform more than 50-fold less well, on a molar basis, than molecules of 40 kb. And the optimal voltage gradient is strongly dependent on the different sized molecules, for instance, pUC19 reaches the highest transformation efficiency at 21 kV/cm, while the 180 kb BAC DNA gets its best efficiency at 13 kV/cm. This paper demonstrates that conditions may be selected which increase the average size of BAC clones generated by electroporation and could be widely applied in large-insert genome library construction.
Chromosomes, Artificial, Bacterial ; genetics ; DNA, Bacterial ; chemistry ; genetics ; Electroporation ; methods ; Escherichia coli ; genetics ; Molecular Weight ; Plasmids ; genetics ; Transformation, Genetic

OBJECTIVETo study the effects of UV irradiation on DNA ligation and transformation efficiency of the expression vector into competent bacterial cells.

METHODSThe expression vector was digested with the restriction enzyme SfiI, and the purified target DNA fragments were exposed to UV light at different wavelengths. Ligation and transformation experiments with the exposed fragments were carried out and the colony number and transformation efficiency were assessed.

RESULTSThe transformation efficiency of the DNA with a 5-min exposure to 302 nm UV was 60 colonies per nanogram of the DNA, as compared with 20400 for the DNA exposed to 365 nm UV. The time course experiment showed that prolonged DNA exposure to 365 nm UV light was associated with lowered transformation efficiency. DNA exposure for 30 min caused a reduction of the transformation efficiency to lower than 50% compared to that of DNA without UV exposure. But with a 15 min exposure, the DNA maintained a transformation efficiency more than 70%, which was sufficient for most molecular biology experiments.

CONCLUSIONIn construction of the expression vector, it is advisable to prevent the target DNA from UV exposure. When UV exposure is essential, we suggest that 365 nm UV be used and the exposure time controlled within 15 min.

Bacteria ; genetics ; DNA Damage ; radiation effects ; DNA Repair ; Genetic Vectors ; radiation effects ; Transformation, Bacterial ; radiation effects ; Ultraviolet Rays

OBJECTIVETo construct a recombinant bacillus Calmette-Guérin vaccine (rBCG) secreting human interferon-alpha 2b (IFN alpha-2b).

METHODSBCG Ag85B signal sequence and IFN alpha-2b gene were amplified from the genome of BCG and of human peripheral blood by polymerase chain reaction (PCR), respectively. IFN alpha-2b gene was cloned in E. coli-BCG shuttle-vector pMV261 to get pMV261-IFN alpha-2b. A new recombinant plasmid pMV261-IFN alpha-2b was constructed by inserting BCG Ag85B signal sequence into pMV261-Ag85B-IFN alpha-2b. Then, BCG was transformed with this recombinant plasmid by electroporation, and designated as rBCG-IFN alpha-2b. The DNA and protein expressions of IFN alpha-2b gene in rBCG were determined by PCR and Western blot respectively. Also the quantity of IFN alpha-2b protein secreted by rBCG in culture supernatants was determined by enzyme linked immunosorbent assay (ELISA).

RESULTSBy partial nucleotide sequencing, the DNA sequences of human IFN alpha-2b and BCG Ag85B were consistent with that in the Gene Bank, and were correctly inserted into the shuttle expression vector pMV261 to construct recombinant plasmid pMV261-Ag85B-IFN alpha-2b. BCG was successfully transformed with this recombinant plasmid by electroporation and the recombinant BCG (rBCG-IFN alpha-2b) was capable of synthesizing and secreting cytokine IFN alpha-2b. The concentration of IFN alpha-2b in culture supernatants was quantified by ELISA and calculated to be approximately 301.45 pg/ml.

CONCLUSIONSRecombinant BCG secreting human IFN alpha-2b (rBCG-IFN alpha-2b) was constructed successfully and the specific IFN alpha-2b protein can be expressed highly and steadily by rBCG vaccine.

BCG Vaccine ; genetics ; immunology ; metabolism ; Gene Expression ; Genetic Vectors ; Humans ; Interferon-alpha ; genetics ; metabolism ; Plasmids ; genetics ; Recombinant Proteins ; Transformation, Bacterial

BCG has been one of the vehicles for multi-recombinant vaccine. However, low transformation efficiency of BCG with plasmid DNA hampered studies involving expression of foreign antigens in BCG. In an effort to determine the optimal conditions, this study was initiated to investigate factors involved in the transformation of BCG with a Mycobacterium-Escherichia coli shuttle vector, pYUB18, by electroporation. Mycobacterium bovis BCG (strain 1173P2) was grown in Middlebrook (M) 7H9 broth containing albumin-dextrose-catalase and 0.05% tween 80, and transformed BCG was grown in M7H10 agar containing kanamycin for counting viable cells. Pretreatment of BCG with 10 mM CaCl2 improved the transformation efficiency, but overnight incubation of BCG with 1% glycine did not. The transformation efficiency in BCG also varied depending on voltage, resistance, and DNA concentration. The maximum transformation efficiency was obtained when the infinity resistance, 12.5 Kv/cm, and 100 ng of DNA were used, and reached 1.4 x 10(5) CFU/microgram of plasmid DNA, which is about 3-100 times greater than those from previous reports. The transformation conditions described in this study, therefore, will give us a better position for employing BCG as a vehicle for developing multi-recombinant vaccines.
Calcium Chloride/pharmacology ; Comparative Study ; DNA/metabolism ; Electrophysiology ; Electroporation* ; Escherichia coli/genetics* ; Genetic Vectors* ; Glycine/pharmacology ; Mycobacterium/genetics* ; Mycobacterium bovis/genetics* ; Osmolar Concentration ; Transformation, Bacterial/physiology* ; Transformation, Bacterial/drug effects

4"-O-isovaleryltransferase gene (ist) was regulated by positive regulatory genes of midecamycin 4"-O-propionyltransferase gene (mpt) in Streptomyces lividans TK24. A BamH I ~8.0 kb fragment from Streptomyces mycarofaciens 1748 was proved that it contained mpt gene and linked with two positive regulatory genes, orf27 and orf28. Orf of mpt was replaced by orf of ist and linked with two regulatory genes or orf27 single, and individually cloned into the vectors pKC1139 or pWHM3 (high copy number), and then transformed into S. lividans TK24. The levels of mpt and ist expression were evaluated by the bio-tramsformation efficacy of spiramycin into 4"-O-acylspiramycins in these transformants. The results showed that 4"-O-isovalerylspiramycins could be detected only in the transformants containing the plasmids constructed with pWHM3. The efficacy of bio-transformation of the transformants containing two regulatory genes was higher than that of orf27 single. So, the positive regulatory genes system of mpt gene could enhance ist gene expression.
Acyltransferases ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; Plasmids ; Spiramycin ; analogs & derivatives ; biosynthesis ; Streptomyces ; enzymology ; genetics ; Streptomyces lividans ; metabolism ; Transformation, Genetic

The dominant gene Xa21 with broad-spectrum and high resistance to Xanthomonas oryzae pv. oryzae (Xoo) was transferred into C418, an important restorer line of japonica hybrid rice in China using double right-border (DRB) T-DNA binary vector through Agrobacterium-mediated transformation. 17 transgenic lines were Xa21-positive with high resistance to the race P6 of Xoo through PCR analysis and resistance identification, among the total 27 independent primary transformants (T0) obtained. The subsequent analysis of the T1 progenies of these 17 T0 lines through PCR-assisted selection and resistance investigation showed that four Xa21 transgenic T0 lines could produce selectable marker-free (SMF) progenies. The frequency of primary transformants producing SMF progenies was 15%. In addition, PCR analysis also revealed these SMF progenies did not contain vector backbone sequence, and they were named as SMF and vector backbone sequence-free (SMF-VBSF) Xa21 transgenic plants. The further molecular and phenotypic analysis of the T2 and T3 progenies testified the homozygous SMF-VBSF Xa21 transgenic plants were obtained with high resistance to Xoo.
DNA, Bacterial ; genetics ; Genetic Vectors ; Oryza ; genetics ; Plant Proteins ; genetics ; Plants, Genetically Modified ; genetics ; Protein-Serine-Threonine Kinases ; genetics ; Rhizobium ; genetics ; Transformation, Genetic ; Xanthomonas

In order to obtain a yeast strain able to produce L-lactic acid under the condition of low pH and high lactate content, one wild acid-resistant yeast strain isolated from natural samples, was found to be able to grow well in YEPD medium (20 g/L glucose, 20 g/L tryptone, 10 g/L yeast extract, adjusted pH 2.5 with lactic acid) without consuming lactic acid. Based on further molecular biological tests, the strain was identified as Candida magnolia. Then, the gene ldhA, encoding a lactate dehydrogenase from Rhizopus oryzae, was cloned into a yeast shuttle vector containing G418 resistance gene. The resultant plasmid pYX212-kanMX-ldhA was introduced into C. magnolia by electroporation method. Subsequently, a recombinant L-lactic acid producing yeast C. magnolia-2 was obtained. The optimum pH of the recombinant yeast is 3.5 for lactic acid production. Moreover, the recombinant strain could grow well and produce lactic acid at pH 2.5. This recombinant yeast strain could be useful for producing L-lactic acid.
Candida ; genetics ; isolation & purification ; metabolism ; Genetic Vectors ; genetics ; L-Lactate Dehydrogenase ; genetics ; metabolism ; Lactic Acid ; biosynthesis ; Metabolic Engineering ; Recombination, Genetic ; Rhizopus ; enzymology ; genetics ; Transformation, Bacterial

OBJECTIVETo establish the transformation system of mulberry, and test its ability of quercetin biosynthesis.

METHODHairy roots of mulberry were obtained through infecting etiolated seedlings with Agrobacterium tumefaciens strain C58C1. The culture condition of hairy roots was optimized. The transformation of T-DNA was examined by PCR assay and quercetin content was determined by HPLC.

RESULTWhen infecting stem cutting of etiolated seedlings via C58C1 strain, the optimal transformation conditions were as follows: 10 minutes' infection, two-days pre-culture and co-culture, additional hydroxylacetosyringone (As) 100 mg x L(-1). The PCR examination result showed that rolB and rolC genes could be inserted into the hairy roots of mulberry. Hairy roots appeared in 10 days after infecting, the frequency of stems explants was up to 92% after 30 days culturing. After 50 days culturing in 1/2MS + 0.05 mg x L(-1) IBA liquid medium, the content of quercetin increased by 8. 5-fold.

CONCLUSIONHairy root culture system of Moraceae plants was established successfully for the first time. In addition, it also provides a foundation for further industrial production of active compounds such as quercetin.

Agrobacterium tumefaciens ; genetics ; metabolism ; Cells, Cultured ; DNA, Bacterial ; genetics ; Gene Targeting ; methods ; Genetic Vectors ; genetics ; metabolism ; Morus ; genetics ; metabolism ; microbiology ; Quercetin ; biosynthesis ; Transformation, Genetic