Homework answers / question archive / Lab Report Recombinant DNA Technology Write-up as a methodology paper:  You can use the following paper as a guide on how to writeup your report “ Simple and efficient site-directed mutagenesis using two single-primer reactions in parallel to generate mutants for protein structure-function studies | BMC Biotechnology | Full Text (biomedcentral

Lab Report Recombinant DNA Technology Write-up as a methodology paper:  You can use the following paper as a guide on how to writeup your report “ Simple and efficient site-directed mutagenesis using two single-primer reactions in parallel to generate mutants for protein structure-function studies | BMC Biotechnology | Full Text (biomedcentral

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Lab Report Recombinant DNA Technology

Write-up as a methodology paper:  You can use the following paper as a guide on how to writeup your report “ Simple and efficient site-directed mutagenesis using two single-primer reactions in parallel to generate mutants for protein structure-function studies | BMC Biotechnology | Full Text (biomedcentral.com)”

Please note that this is a different method than you used in the lab.

Format: Marks will be deducted for missing information and not following directions.

3-4 pages of text excluding Tables, Figures and References.

Times new Roman 12 point, 1.5x spacing

1 inch (2.54cm) margins

Referenced according to a standard JBC (Journal of Biological Chemistry) journal format

Introduction: 3 marks   

Provide some background on the method of site-directed mutagenesis and what the methodology allows in experimental design. Introduce the method, you are writing a proof of principle publication.

Also include some background on TAFI, i.e. function, role in disease, etc?

Note: TAFI is encoded by the CBP2 gene in humans.

M&M: 2 marks 

Refer to the lab manual

In addition, you should provide information on the methods used to translate the DNA, the source of phosphorylation site information, the tools/methodology used to design the primers etc.  RESULTS: 6 marks 

All figures and tables should have a Title and Legend that describes the experiment performed. They should stand on their own in a manner that allows the reader to interpret the data from the information provided. The text within the results should refer to the figures. You state what the results are. You do not interpret the data here.

You should look up some typical articles to get an idea of how to properly convey the information you’re trying to convey. The article noted above will also serve as a guide. 

Things to include here:

Describe your results from the experiment done in the lab.  

Include in your results:  Images from your: Control digests, Site directed mutagenesis PCR diagnostic digests, Plasmid digests, appropriate tables

Look up phosphorylation sites on TAFI. They can be found here: CPB2 (human) (phosphosite.org)

Show all the Phosphorylation sites in a table

1) Note the mutation made in this lab involved mutating the base pair encoding Threonine  (ACT) to Isoleucine (ATT) at amino acid position 346 of TAFI. This results in the loss of the SpeI restriction site.  

The nucleotide sequence of WT TAFI can be found in your lab manual. 

Design: design primers to mutate two independent phosphorylation sites in TAFI that are of unknown function. The mutations should be phosphomimetic converting Ser/Thr to Asp or Glu and Tyr sites to Glu

The Nucleotide and amino acid sequence of TAFI is in the lab manual. You can also translate the FASTA formatted nucleotide sequence of TAFI here:  http://web.expasy.org/translate/   This can be performed as described here:

1. Go to the website: http://www.bioinformatics.org/primerx/
2. Click on the link: ‘enter a mutation in your template DNA sequence’
3. Copy and paste the DNA sequence below in FASTA format into the text area in step 1.
4. Enter the desired mutation
5. Select the appropriate set of primers based on the criteria mentioned above.

You should also include a figure noting the P sites to be mutated i.e. translate the sequence above and display nucleic acid sequence with the protein sequence

NOTE: Alternatively, Agilent also provides a website to design primers that utilizes this method. This can be found here:  QuickChange Primer Design (agilent.com)

Discussion: 4 marks 

You should interpret your results here. If your results are not as expected you may refer to the representative data that will be posted. You should note this issue in your results and include both sets of data if this is the case.  

In the discussion you should comment on how this methodology could be applied in research in regard to the function of these sites on TAFI. You should include the types of experiments that may be performed next to get at various aspect of TAFI function

References: (- 2 marks if incorrect/missing) 

Remember to include in text citations and a reference list.