GFP Protein purification from recombinant vector in E coli cell

GFP Purification from recombinant pQE30 vector inside E.Coli Cell
The abstract
It should be descriptive and specific, essentially “my topic in a nutshell”.
Should be understood separately from the report itself and covers the following: What was done?
Why was it done? How was it done? What was found? What is the significance of the findings?
Introduction
The introduction should present appropriate background material, supported by references. It
should include a statement regarding the major aim/objective(s) of this report.
Experimental Procedures
This report does not warrant a separate section for Materials – describe reagents and apparatus if
needed within the text.
The experimental procedures (Methods) should include all the five major lab modules that were
carried out this semester (you may choose to combine content as you see fit if it is cohesive) as
well as incorporate the methods related to the additional data you have been provided.
Most methods sections are organized under subheadings. If you do use subheadings, make them
functional (e.g., Subcloning the GFP Open Reading Frame) rather than chronological (e.g., Prac 2).
This section should be written in past-tense format and in a concise style to reflect the key
experimental information needed to understand what was done. Use references to key original
citations (e.g., Inoue method for preparing competent cells, Promega Wizard kits for DNA clean up
or minipreps, etc.) but do not reference the practical manual.
Method
1. Cloning of GFP
• PCR amplification GFP open reading frame • Analysis by DNA agarose gel electrophoresis •
Restriction enzyme digestion
2. Restriction enzyme digestions and ligations
• Purification and quantification of digested DNA • Ligation reactions
3. Competent cells and transformation
• Preparation of competent E. coli cells
• Transformation of DNA construct into E. coli cells
4. Cloning confirmation and GFP induction
• Confirmation of clones visually
• Preparation of plasmid DNA from transformed colonies
• Analysis by restriction enzyme digestion and agarose gel electrophoresis
5. Expression and purification of recombinant GFP
• Protein induction and expression in bacteria
• Purification using immobilised metal affinity chromatography
• Analysis by SDS-PAGE
Experimental workflow of GFP Purification from recombinant vector in E.coli.
1.Cloning
Amplification of GFP insert by PCR
Ligation of insert into plasmid vector
Propagation of plasmid
2.Protein expression. Purification of plasmid
Induction of GFP protein expression in bacteria. Restriction analysis
Protein Purification

3.Verification
Verification by SDS-PAGE
Verification by Western blotting
Discussion
In this section, the data should be related back to the literature, ideas presented in the
Introduction and the original aims.
The Discussion should demonstrate a clear understanding of the work through critical analysis and
interpretation of the experimental outcomes and implications. This could include the following
(among others): comparison between observed and expected data – e.g. why was Vent DNA
polymerase used, analysis of PCR product size, ligation reactions, transformation outcomes,
restriction digest analysis to screen for positive colonies); how control reactions were used or
could have been incorporated (e.g. for the PCR amplification of the GFP ORF); how qualitative data
(i.e. SDS-PAGE results) can be enhanced by quantitative protein purification (based on the protein
assays and fold-purification exercise) and qualitative (e.g. Western blot) data acquisition. Any
aberrant data is pointed out and explained logically (if applicable).
You must discuss the additional data provided. Can you probe this data further and rationalize in
the context of your other data? You should discuss the future directions of this work and how
further purification could be achieved. Include at least one modification or addition to the
protocol based on your results which could be incorporated post purification by IMAC and at least
one modification or addition to the protocol which could be incorporated pre purification by
IMAC.