Reference no: EM133238153

Research Design in Molecular Biology and Bioinformatics

For this assignment you are expected to design an SOE-PCR approach to amplify and mutate a Green Fluorescent Protein (GFP) open reading frame (ORF)and clone it in a bacterial expression vector.

Go to your Bioinformatics Assignment folder in D2L and find the file named "Green fluorescent-protein-DNA-sequence.fasta"

Step 1) Create a sequence file in Benchling using this file.

Step 2) Find the green fluorescent protein (GFP) encoding sequence in the file using Benchling. Show the annotation. (Copy paste a screenshot of the annotated sequence in the document)

Step 3) Find the fluorophore site in the protein sequence and annotate it (Copy paste a screen shot of the annotated sequence in the document)

We want you to mutate the fluorophore site by SOE PCR. You will need to mutate the Tyrosine residue of the fluorophore site into a Tryptophanresidue.

Step 4) Show the amino acid and the DNA sequence of the fluorophore for GFP, and show which nucleotides need to be changed (and to which nucleotides you have to change them) to get the tyrosine to tryptophane codon mutation.

Step 5) Which colour would we get after introduction of this point mutation based on the literature and give the reference (one of the original references not just the review paper that is required reading for the course)?
You will need to design a SOE PCR approach and a cloning approach (using Primer 3 plus or Primer blast and Benchling) to mutate the GFP and clone it into the multiple cloning site of the pBHSc custom vector.
Create a DNA sequence by importing the pBHSc-costum-vector.gb. file from your bioinformatics folder in D2L.

Step 6) Locate the multiple cloning site and the HIS-tag (protein sequence with 6 histidines in a row) in the vector. Show a screenshot of the plasmid map and the multiple cloning site with the HIStag location

Youmust design a SOE-PCR approach to mutagenize the fluorophore site of the GFP (Tyrosine to Tryptophane). You can then clone the resulting SOE-PCR fragment into the pBHSc custom vector, so that the GFP open reading frame is fused in frame to the HIS-tag protein sequenceof the vector. It is OK to add extra aminoacids at the N-terminus of the GFP protein sequence to achieve this. You also may have to introduce restriction enzyme recognition sites in your primers.Make sure that we can see the final open reading frame of the fluorescent protein with its amino acid sequence and nucleotide sequence when you show the final constructs in the screen shots.

Step 7) Describe the SOE-PCR primer you will use for this mutagenesis. Show the PCR primer sequences (indicate the restriction enzyme cut site if applicable).

Step 8) Show the locations of the primers in the GFP DNA sequence file in Benchling. (Copy paste a screen shot of the annotated sequence in the document)

Step 9) Give the melting temperatures (Tm's) of all the primers. Also give the GC content, and length of each primer

Step 10) Describe the PCR cycling conditions for the first half product PCR based on the Tm's of your primers. Make sure you include the time of each step in the cycle, and number of cycles.

Step 11) Similar to question 10, describe the PCR cycle conditions of the final SOE-PCR based on the Tm's of your primers.

Step 12) Describe the restriction enzymes you will use to clone the SOE-PCR product into the pBHSc vector.

Step 13) Construct the final SOE-PCR product into a Benchling file. Show the open reading frame.Copy paste a screenshot of the annotated sequence in the document.

Step 14) Clone the fragment into the pBHSc vector using the restriction enzymes you have chosen. Show the open reading frame of the HIS-tag mutant GFP. Copy paste a screenshot of the cloned fragment in the multiple cloning in the document.

Step 15) Create a plasmid map of the final pBHSc vector with the mutated GFP (make sure you included the HIS-tag mutant GFP annotationand copy and paste a screenshot into this document.

Step 16) Create a restriction enzyme digest and virtual agarose gel of your pBHSc vector and with mutated GFP. Copy paste ascreenshot of the virtual digest into this document.

Step 17) Finally,copy and paste a screenshot of your Benchling account directory withall the sequence files you generated into this document.