The project was aimed at isolating strains of E.coli that had two plasmids pGFP and pBCKS incorporated in the cells by the process of electroporation. The mini-project was to isolate a plasmid encoding the coding region for the GFP protein that had a selectable marker, a gene resistant to Ampicillin. The primary objective was to introduce this fragment into a second vector that had Lac polymerase promoter region and a selectable marker that was resistant to Chloramphenicol. When cloned in the correct sequence, the bacterial colonies should be resistant to Chloramphenicol as well as be able to emit green fluorescence under UV light. These strains can be isolated using blotting techniques such as western blotting.
Day 1 morning:
Plasmids pGFP and pBCKS are isolated and electroporated into E.coli competent cells and incubated overnight at 4°C on plates containing Chloramphenicol.
Day 1 afternoon:
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GFP protein expression was induced in E.coli cells by time course of induction method. Cell lysate was prepared at time gaps of zero time point, 30 minutes, 1 hour and 2 hours.
Figure: Images of culture plates of Chlor resistant colonies that emit green fluorescence under UV illumination.
Results of protein concentration in whole cell lysates as determined by Protein assay.
Mean of 2 proteins
Mean of 2 proteins
Discussion: The plates contain IPTG which induces the cells to take up the pGFP and express green fluorescent light emitting protein which can be observed under UV illumination. The plates are read on an automatic plate scanner that also measures the absorbance of the dye binding reagent. The results are interpreted from the above graphs about the volumes of each sample containing 10 µg of proteins.
Day 2 morning: A miniprep is done using the overnight culture of pGFP and pBCKS which are resistant to Ampicillin and Chloramphenicol, respectively. The plasmid DNA sample is electrophoresed on agarose gel to match with known pGFP strand sizes.
Day 2 afternoon: The protein content of the whole cell lysates are examined and observed for protein expression in E.coli. Protein gels are made to undergo SDS-PAGE electrophoresis and electrophoreses after staining with Coomassie blue.
Figure: Images of Coomassie and SDS-PAGE electrophoresis.
Discussion: Coomassie blue is used to stain the lysates in order to find the protein content in gel. In SDS-PAGE the protein content with size similar to that of GFP is checked for any increase in content. The results are obtained after electrophoresis.
Day 3 morning: Restriction enzymes with double digestion of pGFP and pBCKS are made to act on plasmid DNA. DNA obtained after digestion by restriction enzymes are precipitated with ethanol and Agarose gel electrophoresis is performed. pGFP and pBCKS are ligated and electroporation of ligation products is carried out.
The electroporation products are plated on Luria Broth plates that have Chloramphenicol and IPTG, an inducer, incorporated in them.
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Day 3 afternoon: Electrophoresis is carried out using SDS - PAGE, Western blotting techniques.
For this, nitrocellulose sheet in 50mL of 5% skim milk is incubated and left at 4°C until the next practical class.
Western blot is developed; Primary antibody is attached onto the Western blot filter and then the secondary antibody. Colorimetric development of Western blot will be conducted in the next practical class.
Discussion: In Western Blotting technique, the results are interpreted with the reactions to antibodies that are immobilized on the nitrocellulose sheets. However, prior to that, electrophoresis has to be carried out in agar gel and the proteins are then transferred from SDS acrylamide gel onto the nitrocellulose membrane. The results obtained may be compared to the results obtained from Coomassie staining done in the previous week.
In this experiment, plasmid pGFP coding green fluorescence and resistant to Ampicillin and plasmid pBCKS resistant to Chloramphenicol are cloned in the DNA of E.coli. The cDNA of E.coli present in the culture plates is then able to express the green fluorescent light emission as observed under UV illumination as well as show resistance to Chloramphenicol. These are then made to undergo action by double digest restriction enzyme and electrophoresed. Coomassie blue is used to stain the lysates to help demonstrate the results.
Electrophoresis is conducted by passing an electric current through the gel containing cell lysates in wells. The smaller fragments have greater mobility and hence traverse greater distances through the gel mesh whereas the larger strands lag behind. This technique is useful in comparing sizes of fragments with known samples and is widely used in Western and Southern blots as well.
The experiment was successful. However, the results of the Coomassie stain was not very clear due to over-staining. This can be kept under control by paying proper attention while dyeing.
This is a good method of understanding these techniques and carrying out these practical classes help us reinforce what we learn from text books.