Restriction enzymes cut DNA at certain sites to create multiple DNA fragments. Restriction enzyme HindIII has known DNA fragment lengths and recognition sites when digesting lambda DNA, while the lambda DNA recognition site for restriction enzyme XhoI is unknown. The goal of this study is to determine the lambda recognition site of XhoI by comparing a HindIII digest and a HindIII and XhoI double digest on an electrophoresis gel. The HindIII digest had a band at 9.4 kb, but this band was not visible in the double digest, therefore we concluded the recognition site for XhoI was around 9.4kb. There were also two additional DNA
The vital components and techniques of gene cloning are as follows, the DNA sequence that contains the desired gene (EZH2) is amplified by Polymerase chain reaction. PCR was established by Kary Mullis in 1985, popularly known to amplify target sequences of DNA (EZH2) to a billion fold in several hours using thermophilic polymerases (Taq) ,primers and other cofactors (Sambrook and Russell, 2001). Three crucial steps are involved which are Denaturation (at 95°), Annealing of the forward and reverse primers (55-65°) and lastly primer extension (at 72°). After amplification the desired sequence is integrated into the circular vector (pbluescript) forming the recombinant molecule. For the compatibility of the insert and vector, both were digested with (EcoR1) so the same cohesive ends are generated in both, making it easier to ligate. EcoR1 is a restriction enzyme that belongs to the type II endonuclease class which cuts within dsDNA at its recognition site “GAATTC” (Clark 2010; Sambrook and Russell, 2001).
1.) My hypothesis that the insects would be most repelled by the lemon plant extract was not supported. Our key findings in the experiment do not support my hypothesis because the lemon had a 60% repulsion response and was the second most repulsive behind orange with a 70% repulsion response. Although the Earthworms were repelled by the lemon plant extract, they were more so repelled by the orange plant extract. 2.)
The selected isolate Gur1 (6 mm disc) was grown on basal salt media supplemented with 2% carboxymethyl cellulose (CMC) at 28º C until substantial growth was recorded (Hankin and Anagnostaksis 1975). The Petri plates were flooded with congo red solution (0.1%), and after 5 min congo red solution was discarded. The plates were then washed with 1M NaCl solution, and allowed to stand for 15-20 min. The clear zone was observed around the colony when the enzyme had utilized by
Analysis of DNA from practicals 1 and 2 using the technique of agarose gel electrophoresis and analysis of transfomed E. coli from practical 2 (part B)
Genomic DNA was extracted from the fungal mat of Sclerotium rolfsii. Thirty mg of freeze-dried mycelium was ground to a fine powder in an Eppendorf tube in liquid nitrogen. The ground mycelium was resuspended and lysed in 500 µl of lysis buffer (40 mM Tris-acetate, 20 mM sodium acetate,1 mM EDTA, 1% w/v SDS pH 8) (Lerner and Model1981). RNase A (2 µl of 10 mg/ml; Sigma USA) was added and the mixture was incubated for 5 min at 37 °C. To facilitate the precipitation of most polysaccharides, protein and cell debris, 165 ml of 5 mol/l NaCl solution was added and the components mixed by inverting the tube several times. The suspension was centrifuged at 6700 x g for 20 min at 4 °C, the supernatant was immediately transferred to a fresh tube and
Agarose GE: Figure 1 shows the results of amplifying five types Arabidopsis thaliana fwa gene. successful PCR was a result of nice bands showing in figure 1. Wild type undigested (900bp), mutant undigested(860bp), and mutant digested (850bp) were all showing band in the same location. On the other hand, Wild type digested did not show any bands meaning amplification did not occur because McrBc digests fwa enzyme that is being methylated. Gel electrophoresis was used to determine the size of the base pairs from the logarithmic equation. Digested and undigested mutant genes showed hazy regions meaning they have less amount of amplification compared to wild type undigested which shows a significant amplification. No band was shown for
3.1 Cloning, expression and purification of the mutants engineered for rice chitinase allergen: Two residues in each epitope were selected by DNAstar (antigenic index profile) for mutations namely [epitope R1 - arginine18 → tryptophan, aspartic acid22 → methionine; epitope R2 - asparagine35→ methionine, proline41 → isoleucine; epitope R3 - arginine146 → tryptophan, arginine152 → alanine] (Figure 1). Following site-directed mutagenesis, the recombinant clones (per mutant) were sequenced to confirm the mutations. Only the clones with expected mutations were selected for further studies. The mutants were expressed in E. coli cells as fusion proteins with N-terminal 6x histidine tag and were purified to homogeneity yielding
The cloning, expression, and purification of tannase enzyme obtained from bacterium L. plantarum were conducted as described (Wu et al., 2013).
The Ramy3D promoter and 5′ UTR were amplified using PCR reaction from rice genomic DNA. As shown in Figure 1, a 995 bp fragment was obtained from PCR reaction. The product sizes were consistent with our expected length. The amplified fragment was ligated into the pTG19-T vector to obtain pTG19-RamyPro recombinant vector. The pTG19-RamyPro vector was digested with BamH I restriction enzyme to further confirm the cloning of the desired fragment. The digestion reaction showed the correct insertion of the desired fragment into pTG19-T vector (Figure 2).
Antagonistic microorganisms namely Bacillus amyloliquefaciens MB101 (BA) (Solanki et al. 2012), Streptomyces atrovirens N23 (SA) (Malviya et al. 2011) and Hypocrea lixii NAIMCC-F-01760 (HL) (Solanki et al. 2011) were obtained from National Agriculturally Important Microbial Culture Collection (NAIMCC), National Bureau of Agriculturally Important Microorganisms (NBAIM), Mau, Uttar Pradesh, India. Fungal cultures R. solani AG-4 (RS) and HL were maintained on potato dextrose agar (PDA, HiMedia, India) at 25±2°C. Bacterial culture BA was maintained on Nutrient agar (NA, HiMedia, India) at 28±2°C and actinomycetes culture SA on ISP2 media at 28±2°C.
Cellulose is the most abundant compound produced from stalks, leaves, and stems (Shankar, 2011). The use of enzymes in the food industry provides safer and higher quality products. Cellulases are enzymes which break down the sugar cellulose. Cellulase enzymes are produced by fungi, animals, plants, and bacteria. (Zhang 2013) The cellulase enzyme has been used for various industry applications such as the textile industry, paper industry, and juice industry. Cellulases link beta, 1,4 linkages in the cellulose chains (Zhang 2013). There are three types of cellulase enzymes: endoglucanases, exoglucanases, and beta-glucosidases. Exoglucanases act on the reducing or non-reducing ends of cellulose (Zhang, 2013). Endoglucanases cut the nonreducing ends of cellulose or the beta-1,4-bonds. Endoglucanases are also produced by bacteria, fungi, plants, and animals (Zhang,2013). Lastly, beta-glucosidases are produced by archaea, bacteria, fungi, plants, and animals and are known for degrading cellobiose (Zhang, 2013). All three forms of cellulases are produced from animals, bacteria, and plants. The purpose of this paper will be to discuss mainly the cellulase enzyme and its effect on the juice industry in addition to other applications of cellulase, and how cellulase behaves in combination with other enzymes such as pectinase and xylanase.
As mentioned earlier there are three different types of GBSSI enzymes found in wheat- GBSSI-A, GBSSI-B and GBSSI-D. Protein studies have revealed the null mutants of either of these three isoforms. Several studies have been able to identify GBSSI-null A (Shahriary et al., 2012), GBSSI-null B (Demeke et al., 2000), GBSSI-null A+B (Demeke et al., 1997) and GBSSI-null D (Yamamori et al.,