This technique separates Rubisco samples based on their size. The electrophoresis has a positive and a negative end. Positive charge proteins are loaded from the positive end and migrate towards the negative end. Negative charge proteins are loaded from the negative end and migrate towards the positive end (Sakthivel & Palani, 2016). The sample that contained the highest molecular weight of Rubisco will travel the shortest distance on the gel while the protein with the smallest molecular weight will travel the longest distance (Sakthivel & Palani, 2016). The size proportion of each Rubisco molecule correlates with the distance traveled. Rubisco will be in its purest form after running through SDS-page since each technique will increase the purity of the protein. If the salting out, the ion exchange and the SDS-page protein isolation techniques are performed on protein Rubisco, then it is purified and separated by solubility, charge, and size. The rationale of this experiment is to isolate the purest form of Rubisco so that it can perform carbon fixation at an optimal
Figure 1 contains gel electrophoresis for protein samples. The lanes were labeled from 1 to 10 from the right to the left. Lane 1 contained the ladder fragment. Lane 2 contained the filtrate. Lane 3 contained the S1 sample. Lane 4 contained the P1 sample. Lane 5 contained the P1 medium salt sample. Lane 6 contained the P1 high salt sample. Lane 7 contained the S2 sample. Lane 8 contained the P2 sample. Lane 9 contained the P2 medium salt sample. Lane 10 contained the P2 high salt sample.
1. EMG 9 and EMG 26 contain strain _lac-_(I- Z+ Y+) and strain _lac -_ (I+ Z- Y-)respectively.Three genes huddled together on the chromosome are required for two strains of _E.coli_ to utilize lactose.Consisting of three genes, namely, _lacZ_, _lacY_ and _lacA_, the _lac_ operon orderly handles these genes to code specific enzymes necessary for the metabolism of lactose. The genes _lacZ_, _lacY_ and _lacI_ would code for beta-galactosidase, galactosidase permease and _lac_ repressor respectively. Regulation of _lac_ operon is also tight and the operon's negative control is made possible by _lac_ repressor (Hill, 1996). With the presence of _lac_ repressor, _lac_ operon will be deactivated and will
The addition of 5-bromo-4-chloro-3-indoyl-β-thiogalactoside (X-gal) in the agar mix acts as an artifical substrate for the enzyme so it produces blue E.coli colonies when hydrolised by the β-galactosidase meaning those specific colonies do not contain the plasmid with the CIH-1 insert (non recombinant E.coli) (Coventry University 2016). Isopropyl-β-D-thiogalactoside (IPTG) an artifical transcription inducer of the lacZ gene is also added into the agar mix, binding to the repressor gene and inactivating it. Therefore when the CIH-1 gene is incorporated within the plasmid MCS and inserted into the E.coli
Plasmid map of pRSETB. Primers were designed to amplify via PCR to cDNA. The PCR product was digested with XhoI and EcoRI enzymes and ligated into the pRSETB plasmid. The pRSETB plasmid contains a T7 promoter region, is ampicillin resistant, inducible with Isopropyl β-D-1-thiogalactopyranoside IPTG, a molecular mimic of a lactose metabolite that triggers transcription of the lac operon, and has XhoI and EcoRI cut sites. The pRSETB plasmid is transformed into dH5α E. coli and plated on carbenicillin plates. Colonies are selected and grown on a carbenicillin plate while PCR is used to check that the plasmid that was up-taken was not
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).
In analyzing the effect of 2-aminopurine on mutation frequencies of bacterial strain CC102 (ara∆(gpt-lac)5 thi rpsL/F’ lacZ-Y+A+proA+B+ , the bacterial strain CC102 was cultured in LB medium with 2-aminopurine and one LB medium without 2-aminopurine. The bacterial strain CC102 with and without the 2-aminopurine mutagens were diluted to 10-5 and 10-6 and were plated on LB plates with nutrients and sources of carbon compounds for growth. The undiluted bacterial strain CC102 was then plated on minimal medium with thiamine and lactose. In noting the genotype of the bacterial strain, there is a deletion in the lac gene as it is ∆(gpt-lac) and the lacZ gene is not functional, therefore β-galactosidase is not able to cleave lactose into the constituents of glucose and galactose. In table 1.1, when observing the number of mutant colonies on minimal lactose and single cell colonies formed on LB plates with no introduction of the 2-AP mutagen, the mutant colonies were not formed on the minimal lactose yet were formed on the LB nutrient rich plate. For example in looking at group 6 data, 163 colonies were formed on LB medium at 10-6 yet no colonies were formed on minimal lactose with no 2-AP. Since the lac gene and the lacZ gene need to be functional to cleave lactose, it would allow for growth on the minimal medium with lactose but since there was a deletion for the lac gene and lacZ gene was not functional, no growth occurred. However with the introduction of the
For the second part of the experiment, one had to use the knowledge learn from viewing protein molecules in FirstGlance in Jmol to analyze the protein PDB ID: 4EEY. The analysis of this protein was done using the RSCB protein data bank (PDB) at (http://www.rcsb.org/pdb/home/home.do).2
The purpose is to transfer biological samples from a gel to a membrane and their subsequent detection on the surface of the membrane. An antibody is used to specifically detect its antigen for protein analysis. The specificity of the antibody-antigen interaction enables a target protein to be identified in the middle of a complex protein mixture.
In order to manipulate, clone and express certain genes it is essential that a greater understanding of how the gene in question operates, in order to isolate and potentially recreate that particular characteristic in a host organism (Strand ect al. 2003) Escherichia coli is almost the most used organisms of preference in lab based experumentation for the manufacturing of recombinant proteins. Its used as a mobile factory because it is stable and it has become the most famous expression platform organism to use. (Lee ect al.
Through multiple experiments, promoter elements were examined to see how they control gene expression. The purpose was to learn how a gene is regulated in bacteria, how timing affects activity, how positive and negative regulation control gene expression, how E. coli mutants respond in plate assays, and how concentrations of sucrose affects gene expression. ß-galactosidase enzyme, also known as Beta-gal or ß-gal, was the assay in the experiments. ß-galactosidase is an enzyme that breaks up lactose into galactose and glucose. Furthermore, it is a hydrolase enzyme that catalyzes the hydrolysis of ß-galactosides into monosaccharides.
Methods: A pET-expression vector was cloned with various scFv and grown in Escherichia coli bacterial culture. The E.Z.N.A DNA/RNA isolation kit was used for
Spectrophotometer; the finding of protein concentration of an unknown sample of BSA, and by using the standard curve.