The Study Of Fermentas Coli Origame

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).

cloning of plasmid-EZH2 Gene into pBluescript II KS+ plasmid with corresponding Histidine tag for potential analysis

Introduction: Transformation is used to introduce a gene coding for a foreign protein into bacteria. Hydrophobic Interaction Chromatography (HIC) is used to purify the foreign protein. Protein gel electrophoresis is used to check and analyze the pure protein. Research scientists use Green Fluorescent Protein (GFP) as a master or tag to learn about the biology of individual cells and multicultural organisms. This lab introduces a rapid method to purify recombinant GFP using HIC. Once the protein is purified, it may be analyzed using polysaccharide gel electrophoresis (PAGE).

-A common phagemid containing several useful sequences for use in cloning that designed to simplify commonly used cloning and sequencing procedures. It has an extensive polylinker with 21 unique restriction enzyme recognition sites. It has multiple MCS and 21 unique restriction enzyme recognition sites. Also, it has High copy number ColE1-based phagemid, large and versatile polylinker in two orientations; f1 origin available in either (+) or (–) orientation, and T3 and T7 promoters for in vitro transcription of RNA

The purpose of the experiment was to isolate plasmid DNA, followed by restriction digestion using restriction endonucleases and then visualizing the digested fragments after subjecting to gel electrophoresis. Plasmid DNA (pSP72 DNA) was isolated from Escherichia coli KAM32 (E.coli) cultures using the QIA prep miniprep kit and then subjected to restriction digestion by EcoRI and HindIII. The restriction digested DNA was then loaded into the wells of 0.7% agarose gel and subjected to electrophoresis. It can be concluded from our results that our plasmid DNA isolation was successful and the restriction digestion results were partially in agreement with our hypothesis.

The pET30a expression vector featuring T7 promoter, N/C-terminal His-tag sequence, T7 terminator and Lac operator was used for ligation of F protein gene at EcoRI restriction sites. The F gene-pET30a ligation was transformed into E.coli strain TOP10 competent cells and spread on Luria Bertani (LB) plates with kanamycin selection. The single colonies were picked and screened for positive cloning through EcoRI restriction digestion. The correct orientation of the gene was confirmed through restriction digestion of the PCR product with ScaI and EcoRV.

The experiment was conducted in two main parts: in the first part, the single and double restriction digests were run to cut the plasmids using combinations of three different enzymes. In the second part, the fragments resulting from each reaction were separated using gel electrophoresis. 1) Restriction Digests In this part of the experiment, seven different samples were prepared with different combination of reactions. Given the unknown plasmid and three restriction enzyme (HindIII, BglI and PvuII), seven different Eppendorf tubes were prepared to account for 3 single digests, one with each of the three restriction enzymes, 3 double digests combining two enzymes at a time and alternating between all the possible combinations, and also for the control reaction where no enzymes were added to the plasmid.

Tachylectin-5A is an ellipsoidal molecule that is an asymmetrical monomer, which is subdivided into three different domains that interact with each other (2). The primary structure of Tachylectin-5A constitutes 269 amino acid residues (1). The amino acid composition of this protein has a cis-peptide bone between Arg-218 and Cys-219 (3). It contains one protein modification called prryolidone carboxylic acid, which is the twenty-fourth amino acid in the sequence (1). And the secondary structure of the protein consists of 16% alpha helical and 24% beta sheets. There are 9 helices with 44 amino acid residues and 19 beta sheets with 66 residues involved in total (1). The

In order to clone and express the luciferase gene in E.Coli, first, isolation of the chromosomal DNA from V. Fischeri with phenol extraction, along with purification, is necessary before amplification through Polymerase Chain Reaction (PCR) [1].

[5]Fernandez-Gomez, Francisco J., Maria F. Galindo, Maria Gómez-Lázaro, Victor J. Yuste, Joan X. Comella, Norberto Aguirre, and Joaquín Jordán. "Abstract." National Center for Biotechnology Information. U.S. National Library of Medicine, 17 Jan. 2005. Web. 08 Oct. 2014. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1576031/#bib3>.

The Lambda kit was used to rid the digested pGEM of the stuffer fragment so as to lower the probability of it re-ligating back into the plasmid instead of the luxAB inserts [1]. In total, four ligation reactions were prepared under varying conditions – L1 contained a 1:1 insert to plasmid (with stuffer) ratio, L2 contained a 2:1 insert to plasmid (with stuffer) ratio, L3 contained a 1:1 insert to plasmid (no stuffer) ratio, and L4 contained a 2:1 insert to plasmid (no stuffer) ratio. Strains of highly competent Zippy cells, derived from E. coli DH5 cells, were diluted and transformed with the recombinant plasmids [1]. pGEM contained the Ampr gene which inhibited ampicillin via -lactamase, thus, DH5 cells that took up pGEM were able to actually grow in the agar plates containing ampicillin [1]. In order to determine the efficiency of the ligations and observe any activity of luciferase, several transformations were plated, and the produced blue and white colonies were counted [1]. If pGEM contains an insert, the insert itself will inactivate LacZ by disrupting its sequence -- blue products result from intact pGEM because -galactosidase would properly cleave X-gal, and white products result from cut pGEM because -galactosidase would be non-functional [1]. The negative control was comprised of an aliquot of DH5 cells and sterile water, the positive control was

Gene BglA was sub-cloned in pET21a (+), recombinant vector transformed and propagated in E. coli BL21 CodonPlus under the control of T7/lac promoter system. The positive bacterial transformants were grown in LB-ampicillin broth (100 µg mL−1) and induced with IPTG for the production of recombinant BglA enzyme. Optimal heterologous expression and efficient folding of BglA protein was observed with 0.5 mM IPTG, after 72 h incubation at 22°C. The use of reduced concentration of IPTG for getting maximum gene expression, was proved an efficient method for the production of active soluble enzyme. The protein was purified by applying two subsequent purification steps employing ion exchange and hydrophobic interaction chromatography after heat-treatment

Two Polymerase Chain Reactions (PCR) were set up, one was digested with the restriction enzyme and the other served as the control. 11.0 µl of the forward primer solution, 11.0 µl of the reverse primer solution, and 3.0 µl of the

An important aspect to producing synthesized EPO is the use of mammalian cells. EPO contains 40% carbohydrates which are important to the stability and biosynthesis of recombinant EPO (5). In 1984, EPO was able to be cloned and synthesized for use by using recombinant DNA technology (1, 3, 4, 5). The biological properties of synthesized EPO are very similar to the original hormone found naturally in the body (3). The importance of the mammalian cell carbohydrate composition allowed for the carbohydrate portions of recombinant EPO to be almost identical to the carbohydrate portions of native EPO (3). Success in creating synthetic EPO on a large scale that mimics native EPO in humans allowed for the implementation of synthesized EPO for medicinal purposes.

Plasmid DNA with Restriction Digest: The purpose of restriction digest of plasmid DNA is to understand how each DNA plasmids was cut with the given restriction enzymes and perform gel electrophoresis to observe the samples. Nine restriction digests were created, containing three digests for each of the three plasmid DNAs identifying as recombinant, non-recombinant, and unknown. Out of the nine digests, six are actual digests and three are undigested controls. A master mix is created to add to each of the nine samples with its following stock ingredients: 10 ul of 2X Reaction Buffer, 1 ul of Nco1, X ul of sterile water (Single digest), 10 ul of 2X Reaction Buffer, 10 ul plasmid DNA, 1 ul Nco1, 1 ul of Not1, and X ul of sterile water (Double