Participation Traders Separating Waste In Pasar Baru Tampan Sub District Pekanbaru City

Garbage is defined as something that is not used anymore, unused, or something that is thrown away, which is derived from human activities and does not happen by itself. The market is one of the human activities that produce large amounts of garbage every day, when the waste sorting system is not good, it will make it difficult to carry out waste management and will have an impact on health directly or indirectly. This study aims to determine the factors associated with the participation of traders in waste sorting in Tampan Sub district  Pasar Baru  Pekanbaru.This study is quantitative research with cross sectional design. This research was conducted April 2015, sample in this study is 79 merchants. Data were collected by using questionnaires and observation. Data analysis for bivariate with chi-square test with 95% confidence level with α = 0.05. The results showed that there is a relationship between education (OR = 2,60  ; CI: 1,08-3,67),  socialization  (OR = 3,10; CI: 2,58-5,99) availability of trash waste (OR = 8,25 ; CI: 2,98-7,55 with waste sorting participation

Tagging the expressed protein with 6 histidines: rapid cloning of an amplicon with three options.

We report the designing of three expression vectors that can be used for rapid cloning of any blunt-end DNA segment. Only a single set of oligonucleotides are required to perform the amplification of the target DNA and its cloning in all three vectors simultaneously. The DNA thus cloned can express a protein either with or without a hexa-histidine tag depending upon the vector used. The expression occurs from T7 promoter when transformed into E. coli BL21(DE3). Two of the three plasmids have been designed to provide the expressed protein with either N- or C-terminus 6 histidine amino acids in tandem. The third plasmid, however, does not add any tag to the expressed protein. The cloning is achieved quickly with the requirement of phosphorylation of PCR product without any restriction digestion. Additionally, the generated clones can be confirmed with a single step PCR reaction carried out from bacterial colonies (generally termed as "colony PCR"). We show the cloning, expression and purification of Green Fluorescent Protein (GFP) as proof-of-concept. Additionally, we also show the cloning and expression of four sigma factors from Mycobacterium tuberculosis further demonstrating the utility of the designed plasmids. We strongly believe that the vectors and the strategy that we have developed will facilitate the rapid cloning and expression of any gene in E. coli BL21(DE3) with or without a hexa-histidine tag

Diagrammatic representation of the cloning of a PCR amplified DNA segment into the three vectors.

<p>The amplified product should contain the ATG. The blunt-end DNA segment can be cloned in all the three vectors.</p

Sequence of a region of pSK01-NCHS showing the SDM primer binding sites.

<p>Italicized letters represent the start and stop codon. Bold faced letters depict restriction enzyme sites, NcoI, NdeI and XhoI, which were mutated to SmaI.</p

Primers used in this study. SmaI site, wherever present, has been underlined. The forward primers for the cloning of genes start with ATG (boldfaced).

<p>Primers used in this study. SmaI site, wherever present, has been underlined. The forward primers for the cloning of genes start with ATG (boldfaced).</p

Molecular Dissection of the Homotrimeric Sliding Clamp of T4 Phage: Two Domains of a Subunit Display Asymmetric Characteristics

Sliding clamp proteins are circular dimers or trimers that encircle DNA and serve as processivity factors during DNA replication. Their presence in all the three domains of life and in bacteriophages clearly indicates their high level of significance. T4 gp45, besides functioning as the DNA polymerase processivity factor, also moonlights as the late promoter transcription determinant. Here we report a detailed biophysical analysis of gp45. The chemical denaturation of gp45 probed by circular dichroism spectroscopy, tryptophan fluorescence anisotropy, and blue-native polyacrylamide gel electrophoresis suggests that the protein follows a three-state denaturation profile and displays an intermediate molten globule-like state. The three-state transition was found to be the result of the sequential unfolding of the two domains, the N-terminal domain (NTD) and the C-terminal domain (CTD), of gp45. The experiments involving Trp fluorescence quenching by acrylamide demonstrate that the CTD undergoes substantial changes in conformation during formation of the intermediate state. Further biophysical dissection of the individual domain reveals contrasting properties of the two domains. The NTD unfolds at low urea concentrations and is also susceptible to protease cleavage, whereas the CTD resists urea-mediated denaturation and is not amenable to protease digestion even at higher urea concentrations. These experiments allow us to conclude that the two domains of gp45 differ in their dynamics. While the CTD shows stability and rigidity, we find that the NTD is unstable and flexible. We believe that the asymmetric characteristics of the two domains and the interface they form hold significance in gp45 structure and function

Expression analysis of <i>M. tuberculosis</i> sigma factors.

<p>All sigma factors cloned in pMS-QS vectors were expressed in <i>E. coli</i> BL21(DE3) and were analyzed on a 15% SDS-PAGE gel. The gel images show protein profiles in the presence (+) and absence (−) of IPTG. Arrow points to the protein produced upon addition of IPTG only. Panels A, B, C and D correspond to SigB, SigF, SigG and SigD respectively. “M” represents protein molecular weight marker.</p

GFP expression from pMS-QS vectors.

<p>(A) GFP induction profile with all the three vectors. Both uninduced (−IPTG) and induced (+IPTG) cultures have been compared. Arrow depicts the induced GFP expression. (B) Zymogram of A that was imaged using 480 nm light and SYBR Gold filter. The arrow represents the folded GFP protein in polyacrylamide gel that fluoresces upon excitation with 480 nm light. Pre-stained protein marker was used in order to make it appear on zymogram. Purification of GFP protein using Ni-NTA matrix was done after overexpressing the protein in the bacterial cells having pMS-QS-CHGFP vector expressing C-ter GFP (C) and pMS-QS-NHGFP vector expressing N-ter GFP (D). The arrow shows the purified protein band. Only elutions were loaded for analysis.</p

Screening of GFP positive clones using colony PCR.

<p>Agarose gel images after colony PCR showing, (A) clone-3 to be positive in pMS-QS-NOHGFP; (B) clone-1 to be positive in pMS-QS-CHGFP and (C) clones-2, 3 & 4 to be positive in pMS-QS-NHGFP. Vector specific forward primer (PETFor) and gene specific reverse primer (GFPRev-rapid) were used to carry out colony PCR in order to confirm the insertion as well as the orientation of the GFP gene in plasmid.</p

Plasmids used in this study. All the plasmids carry Ampicillin resistance marker and the cloned gene expresses from a T7 promoter, when an appropriate host is used.

<p>Plasmids used in this study. All the plasmids carry Ampicillin resistance marker and the cloned gene expresses from a T7 promoter, when an appropriate host is used.</p