Small Amplicons Mutation Library for Vaccine Screening by Error-Prone Polymerase Chain Reaction

Library construction is a common method used to screen target genes in molecular biology.Most library constructions are not suitable for a small DNA library(<100 base pair(bp))and low RNA library output.To maximize the library’s complexity,error-prone polymerase chain reaction(PCR)was used to increase the base mutation rate.After introducing the DNA fragments into the competent cell,the library complexity could reach 109.Library mutation rate increased exponentially with the dilution and amplification of error-prone PCR.The error-prone PCR conditions were optimized including deoxyribonucleotide triphosphate(dNTP)concentration,Mn^(2+)concentration,Mg^(2+)concentration,PCR cycle number,and primer length.Then,a RNA library with high complexity can be obtained by in vitro transcription to meet most molecular biological screening requirements,and can also be used for mRNA vaccine screening.

Cloning of Bt cry Genes by Rapid Screening of DNA Libraries with PCR-RFLP

Bacillus thuringiensis (Bt) strain C002 contains crylAa, cry2Ab, crylCa insecticidal crystal genes and an unkown gene cryX, among which crylCa is located in a 6 -9 kb EcoR I fragment of the chromosomal DNA. The total DNA and the plasmids DNA libraries of C002 were constructed in Bt-E. coli shuttle plasmid pHT315 by inserting 6 - 9 kb chromosomal and plasmid DNA fragments prepared respectively with EcoR I complete and 5au3A I partial digestion. On the basis of every 50 transformants pooled together from 5-10 tubes, the pools containing about 2 000 transformants from the plasmids DNA library and 400 transformants from the total DNA library were rapidly screened by PCR-RFLP. Clones containing crylAa, cryX, crylCa , and cry2Ab were isolated and named as pHT-1Aa, pHT-X, pHT-1Ca and pHT-2Ab respectively. Restriction analysis indicated that pHT-1Aa, pHT-1Ca and pHT-2Ab had the typical physical map of the homologous cry genes. Furthermore, each plasmid was transferred into Bt acrystalliferous strain cryB- by eletropora-tion. SDS-PAGE result showed that transformant of pHT-1Ca expressed 130 kDa protein and bioassay result proved its high toxicity against Spodotera exigua 1st instar larvae with 100% corrected motality.

Functional Metagenomics from the Rumen Environment—A Review

The rumen microbiome plays an essential role in ruminant physiology, nutrition and pathology as well as host immunity. A better understanding of rumen microbial processes and identification of which populations are responsible for specific functions within the rumen microbiome will lead to better management and sustainable utilization of the available feed base while maintaining a low environmental impact. Recent advance in the culture independent method of microbiology such as metagenomics, unravels potentially the rumen microbial process. There are two basic types of metagenomics studies: Sequence-based and function-based metagenomics. Sequence-based metagenomics involves sequencing and analysis of DNA from environmental samples. Its purpose is to assemble genomes, identify genes, find complete metabolic pathways, and compare organisms of different communities. Whereas functional metagenomics is the study of the collective genome of a microbial community by expressing it in a foreign host usually Escherichia coli (E. coli). It is a promising approach unearthing novel enzymes even from yet to culture rumen microbiota. Further advances in the screening techniques promise vast opportunities to rumen microbiologists, and animal nutritionist. The identification of novel enzyme through functional metagenomics consists of three parts: rumen sample collection;DNA library construction and screening of individual clone. Functional metagenomics was successfully applied to identify different antibiotics, hydrolytic enzymes, antibiotic resistance genes, and many other functions;moreover, it allowed characterization of genes encoding enzymes with a particular activity, which represents completely novel sequence. There are a number of outputs from functionally screened rumen product such as carbohydrate active enzymes (CAZymes) that can break down plant cell walls. Company involved commercialization of metagenomics research such as Syngenta, Genencor International, BRAIN etc., has produced many biological molecules of commercial interest. The aim of this paper is to elucidate functional metagenomics, from rumen environment and its potential for commercial purpose.

Identification of Festuca arundinacea Schreb Cat1 Catalase Gene and Analysis of its Expression Under Abiotic Stresses

Ablotlc stresses, such as drought, high salinity, and cold/freezing, lead plants to produce excess reactive oxygen species. Catalase, a unique hydrogen peroxide-scavenging enzyme, plays a very Important role In plants. To characterize the catalase involved In plant response to ablotlc stresses, we constructed a cDNA library from 4℃-treated Festuca arundinacea Schreb seedlings and isolated a catalase gene from this library. The cDNA （FaCat1, 1 735 bp） contained an open reading frame of 1 479 bp. BLAST analysis Indicated that the deduced amino acid sequence showed 96% Identity with that from wheat TaCat1 and 87% Identity with that from maize ZmCat2. Northern blotting analysis showed an obvious Increase of FaCat1 transcripts In leaves In contrast with roots. Time-course analysis of the expression of FaCat1 in F. arundinacea leaves showed that FaCat1 expression was upregulated in cold- and salt-stressed leaves, with the FaCat1 transcripts accumulat-Ing mostly at 4 or 2 h after cold or salt stress, respectively. No significant changes in FaCat1 transcription were observed in dried leaves and inhibition of FaCat1 transcription was found In absclsic acid （ABA）-treated leaves, Indicating that the FaCat1 gene is differentially expressed during cold, high salt, drought, and ABA treatment In F. arundinacea leaves.

Microbial community structures in an integrated two-phase anaerobic bioreactor fed by fruit vegetable wastes and wheat straw

The microbial community structures in an integrated two-phase anaerobic reactor（ITPAR）were investigated by 16 S r DNA clone library technology. The 75 L reactor was designed with a 25 L rotating acidogenic unit at the top and a 50 L conventional upflow methanogenic unit at the bottom, with a recirculation connected to the two units. The reactor had been operated for 21 stages to co-digest fruit/vegetable wastes and wheat straw, which showed a very good biogas production and decomposition of cellulosic materials. The results showed that many kinds of cellulose and glycan decomposition bacteria related with Bacteroidales,Clostridiales and Syntrophobacterales were dominated in the reactor, with more bacteria community diversities in the acidogenic unit. The methanogens were mostly related with Methanosaeta, Methanosarcina, Methanoculleus, Methanospirillum and Methanobacterium; the predominating genus Methanosaeta, accounting for 40.5%, 54.2%, 73.6% and 78.7% in four samples from top to bottom, indicated a major methanogenesis pathway by acetoclastic methanogenesis in the methanogenic unit. The beta diversity indexes illustrated a more similar distribution of bacterial communities than that of methanogens between acidogenic unit and methanogenic unit. The differentiation of methanogenic community composition in two phases, as well as pH values and volatile fatty acid（VFA） concentrations confirmed the phase separation of the ITPAR. Overall, the results of this study demonstrated that the special designing of ITPAR maintained a sufficient number of methanogens, more diverse communities and stronger syntrophic associations among microorganisms, which made two phase anaerobic digestion of cellulosic materials more efficient.

Continuous desulfurization and bacterial community structure of an integrated bioreactor developed to treat SO_2 from a gas stream

Sulfide dioxide（SO2） is often released during the combustion processes of fossil fuels. An integrated bioreactor with two sections, namely, a suspended zone（SZ） and immobilized zone（IZ）, was applied to treat SO2 for 6 months. Sampling ports were set in both sections to investigate the performance and microbial characteristics of the integrated bioreactor. SO2 was effectively removed by the synergistic effect of the SZ and IZ, and more than 85%removal efficiency was achieved at steady state. The average elimination capacity of SO2 in the bioreactor was 2.80 g/（m3·hr） for the SZ and 1.50 g/（m3· hr） for the IZ. Most SO2 was eliminated in the SZ. The liquid level of the SZ and the water content ratio of the packing material in the IZ affected SO2 removal efficiency. The SZ served a key function not only in SO2 elimination, but also in moisture maintenance for the IZ. The desired water content in IZ could be feasibly maintained without any additional pre-humidification facilities. Clone libraries of 16 S r DNA directly amplified from the DNA of each sample were constructed and sequenced to analyze the community composition and diversity in the individual zones.The desulfurization bacteria dominated both zones. Paenibacillus sp. was present in both zones, whereas Ralstonia sp. existed only in the SZ. The transfer of SO2 to the SZ involved dissolution in the nutrient solution and biodegradation by the sulfur-oxidizing bacteria.This work presents a potential biological treatment method for waste gases containing hydrophilic compounds.