6. In lab, you amplified the GFP coding sequence by PCR as a XhoI fragment, in o
ID: 268318 • Letter: 6
Question
6. In lab, you amplified the GFP coding sequence by PCR as a XhoI fragment, in order to clone it into the XhoI site of the vector pSK.
The GFP insert was amplified in such a way that, after ligation, the GFP coding sequence was in frame with LacZ? present on the plasmid, thereby producing a construct expressing a LacZ?-GFP fusion protein in E. coli.
Your task is to design the 5’ oligonucleotide used to amplify GFP in order to obtain the correct PCR product to clone into pSK (assume you have the 3’ oligonucleotide available). Your 5’ oligonucleotide needs a XhoI site, which, after restriction, will produce an overhang that can be ligated in frame into the pSK vector.
You will need to write down the sequence of your 5’ oligonucleotide as follows:
1) the restriction site (6 nucleotides)
2) any nucleotide you may find necessary to include to preserve the frame (0,1 or 2 nucleotides)
3) 5 codons corresponding to the relevant GFP coding sequence, starting from the second codon of GFP (15 nucleotides).
This will make a sequence of 21 nucleotides, plus 1 or 2 nt if you decide to add them to your sequence in order to preserve the frame. As a side note: 4-6 nucleotides are usually added to the 5’ end in order to provide enough space for the enzyme to cut- we won’t worry about this here.
For this exercise, you need the following information:
•XhoI restriction site: CTCGAG
•The 5’ end of the GFP coding sequence (start codon is underlined):
5’ ATG AGT AAA GGA GAA GAA CTT TT CACTGGAGT TGTCCCAATT CTTGTTGAAT TAGATGGTGA TGTTAATGGGCACAAATTTT CTGTCAGTGG AGAGGGTGAA GGTGATGCAA CATACGGAAA ACTTACCCTT 3’
•The sequence of LacZ? on the vector, including the start codon (underlined) and the XhoI site:
•Clearly indicate the sequence of your oligonucleotide, 5’ to 3’, as per the instructions above. Add your sequence to the nucleotides written below. These four nucleotides are added to the 5’ end of your primer in order to allow for efficient cutting by the XhoI enzyme close to the end of your oligonucleotide.
[ optional: For more information on this, go to this link:
https://www.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments ]
5’ A T G A …
On your sequence:
underline the XhoI site
state clearly whether or not you are adding nucleotide(s), and if you do, circle it (them).
indicate the predicted amino acids (3 letter code) produced at the fusion point: start with the LacZ? residues Ser-Ile-Pro and continue from there into your GFP sequence indicating the 5 residues encoded from the GFP coding sequence.
Explanation / Answer
In this question you have to make primer that contain the Xho I resction site. For making primer take the few base pairs of the GFP nucleotide sequence.
5’ATG AGT AAA GGA GAA GAA CTT 3’
Protein sequence of this segment is
MSKGEEL
Now at the 5’ end add CTCGAG sequence.
5’ CTCGAG ATG AGT AAA GGA GAA GAA CTT 3’
Now add 4 bases for proper functioning of Xho I enzyme.
5’ GGCC CTCGAG ATG AGT AAA GGA GAA GAA CTT 3’
5’ GGCC CTCGAG ATG AGT AAA G 3’
Now here you can reduce the base pair from 3’ end and keep a length up to 20 bp.
After digestion this will ligate the nucleotide sequence of lac z-alpha. Now this time Xho I restriction site again create and new sequence will contain two additional amino acids that come from the Xho I restriction site. Now new sequence that having two additional amino acids tah is Leucine (L) and glutamic acid ( E ).
LEMSKGEEL
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