Socio-Economic Profiling of Members of Women’s Groups in Raipur District of Chhattisgarh

In India, collective farming by Women’s Groups (WGs) was introduced as a strategy to improve needy women’s livelihood alternatives. The study was performed in Raipur district of Chhattisgarh. Three villages were chosen from two selected blocks viz.,Arang and Dharsiwa, and five women groups were selected from each village. Most of the women members of women’s groups were in the middle age category. More than one-third of the women members had education up to middle school level. About fifty percent of the women members had medium family size and almost ninety-six percent of the respondents were married. Nearly 70 percent of the women member of women’s  groups had marginal (<2.5 acres) landholdings. Sixty percent of the respondents had medium level of annual family income. Nearly sixty-eight percent of women members did not receive any training. Majority of the members of women’s groups had medium level of participation in extension and mass media activities.

A novel helper phage enabling construction of genome-scale ORF-enriched phage display libraries.

Phagemid-based expression of cloned genes fused to the gIIIP coding sequence and rescue using helper phages, such as VCSM13, has been used extensively for constructing large antibody phage display libraries. However, for randomly primed cDNA and gene fragment libraries, this system encounters reading frame problems wherein only one of 18 phages display the translated foreign peptide/protein fused to phagemid-encoded gIIIP. The elimination of phages carrying out-of-frame inserts is vital in order to improve the quality of phage display libraries. In this study, we designed a novel helper phage, AGM13, which carries trypsin-sensitive sites within the linker regions of gIIIP. This renders the phage highly sensitive to trypsin digestion, which abolishes its infectivity. For open reading frame (ORF) selection, the phagemid-borne phages are rescued using AGM13, so that clones with in-frame inserts express fusion proteins with phagemid-encoded trypsin-resistant gIIIP, which becomes incorporated into the phages along with a few copies of AGM13-encoded trypsin-sensitive gIIIP. In contrast, clones with out-of-frame inserts produce phages carrying only AGM13-encoded trypsin-sensitive gIIIP. Trypsin treatment of the phage population renders the phages with out-of-frame inserts non-infectious, whereas phages carrying in-frame inserts remain fully infectious and can hence be enriched by infection. This strategy was applied efficiently at a genome scale to generate an ORF-enriched whole genome fragment library from Mycobacterium tuberculosis, in which nearly 100% of the clones carried in-frame inserts after selection. The ORF-enriched libraries were successfully used for identification of linear and conformational epitopes for monoclonal antibodies specific to mycobacterial proteins

Rapid Restriction Enzyme-Free Cloning of PCR Products: A High-Throughput Method Applicable for Library Construction

<div><p>Herein, we describe a novel cloning strategy for PCR-amplified DNA which employs the type IIs restriction endonuclease BsaI to create a linearized vector with four base-long 5′-overhangs, and T4 DNA polymerase treatment of the insert in presence of a single dNTP to create vector-compatible four base-long overhangs. Notably, the insert preparation does not require any restriction enzyme treatment. The BsaI sites in the vector are oriented in such a manner that upon digestion with BsaI, a stuffer sequence along with both BsaI recognition sequences is removed. The sequence of the four base-long overhangs produced by BsaI cleavage were designed to be non-palindromic, non-compatible to each other. Therefore, only ligation of an insert carrying compatible ends allows directional cloning of the insert to the vector to generate a recombinant without recreating the BsaI sites. We also developed rapid protocols for insert preparation and cloning, by which the entire process from PCR to transformation can be completed in 6–8 h and DNA fragments ranging in size from 200 to 2200 bp can be cloned with equal efficiencies. One protocol uses a single tube for insert preparation if amplification is performed using polymerases with low 3′-exonuclease activity. The other protocol is compatible with any thermostable polymerase, including those with high 3′-exonuclease activity, and does not significantly increase the time required for cloning. The suitability of this method for high-throughput cloning was demonstrated by cloning batches of 24 PCR products with nearly 100% efficiency. The cloning strategy is also suitable for high efficiency cloning and was used to construct large libraries comprising more than 10⁸ clones/µg vector. Additionally, based on this strategy, a variety of vectors were constructed for the expression of proteins in <i>E. coli,</i> enabling large number of different clones to be rapidly generated.</p></div

Diagrammatic representation of pVLExp4037/4038/4039 vectors.

<p>Only relevant genes and restriction sites are shown. The maps are not to scale. T7<i>lac</i>, T7 promoter lac operator; RBS ribosome-binding site; T7Tn, T7 transcription terminator; <i>fori</i>, origin of replication of filamentous phage; Amp^r, β lactamase gene; Ori, Col E1 origin of replication; <i>laci</i>, lac repressor encoding gene rop/rom; Stuffer, 1.8 kbp stuffer flanked by BsaI sites. The encoded amino acids are shown in single letter code (bold) below the nucleotide sequence (A–E). F, shows the summary of vectors containing different fusion tags and protease sites.</p

Cloning strategy.

<p>The vector contains two appropriately oriented BsaI sites (A) upon digestion with BsaI linearized vector is obtained with ends having 4-base 5′-overhangs (B) shown in red. The recognition sequence of restriction enzyme BsaI are underlined and the cleavage site is marked. The Gene Of Interest (GOI) is amplified using two gene-specific primers with 7-base long additional sequence at the 5′ end (C) shown in bold. Treatment of PCR product with T4 DNA polymerase and dTTP produces two different four-base overhangs that are complementary to two ends of the linearized vector shown in red (D). The ligation results in direction cloning of the insert into the vector (E).</p

Cloning efficiency of four genes using column method and single tube method.

a<p>The competent cells [BL21(DE3)RIL] used had efficiency of 1×10⁸ transformants/µg supercoiled pGEM3Z DNA.</p><p>Cloning efficiency of four genes using column method and single tube method.</p

Flowchart and timing of the experimental steps for “Time Saver” cloning strategy.

<p>Flowchart and timing of the experimental steps for “Time Saver” cloning strategy.</p

Alignment of the fragments of MTBLIB27 library selected by the monoclonal antibodies (A) Ag85-12 and (B) 1912.

<p>The translated regions of Ag85A, Ag85B and 19-kDa antigens of <i>M. tuberculosis</i> are represented as solid bar. The location of the deduced peptide sequences displayed on affinity-selected phages along with the position of the coded peptide is shown in bold on the right of the corresponding clone bar. The minimal overlapping sequence representing the putative epitope is shown below the alignment of each group.</p