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Experiment 2: Cloning a DNA Fragment to a Bacterially-Derived Plasmid Vector Bacteria frequently contain extrachromosomal plasmids,...

Experiment 2: Cloning a DNA Fragment to a Bacterially-Derived Plasmid Vector

Bacteria frequently contain extrachromosomal plasmids, which are circular DNA genetic elements that are self-replicating. Plasmids are typically not necessary for the bacteria's survival but can sometimes confer a growth advantage to the bacteria. Plasmids can be transferred between bacteria and recombinant DNA technology makes use of this feature to introduce “foreign” genes into bacteria and use the bacteria as a cell “factory” to produce the gene. Genes are inserted into plasmids (also called vectors) by means of restriction enzymes. Restriction enzymes are bacterially derived enzymes that recognize specific DNA sequences and cut (or restrict) that particular DNA sequence in a consistent way. That is, any DNA that contains a particular restriction enzyme sequence will be cut the same. Restriction enzymes often produce overhanging ends of DNA (called sticky ends) that can adhere to each other through hydrogen bonding then DNA ligase permanently links the pieces together by forming covalent bonds between the phosphate backbones of the DNA, creating a recombinant or genetically engineered DNA. In this experiment, you will be given two sequences of DNA: one sequence is a foreign gene and the other sequence is of a plasmid vector. You will use a computer program to identify where the common restriction sites for both sequences occur. Scientists perform this type of computer simulation prior to performing restriction enzyme digestion to ensure that they will cut the sequences as expected.

Materials

NEB Cutter Website: http://tools.neb.com/NEBcutter2/index.php
Computer with Internet access.
Foreign Gene Sequence (provided in experimental procedure)
Plasmid Gene Sequence (provided in experimental procedure)

Procedure

Type the link listed in the materials box for the NEB Cutter website into a web browser (note, cutting and pasting the link from a PDF file format will not work. You must manually type in the address).

Copy and paste the foreign DNA sequence into the box on the NEB Cutter website where it says “Paste In Your DNA Sequence”. Foreign DNA Sequence:

GAATTCGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGT

CGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGC

GAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGG

CAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGC

AGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCG

CCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCA

ACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCA

TCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGC

TGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAAC

GGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGC

TCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCC

CGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGC

GCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATG

GACGAGCTGTACAAGGGATCC

Title the sequence as “Foreign DNA” in the “Name of Sequence” box. Leave the other settings on their default status.

Click the “Submit” button (located to the right of the pasted DNA sequence).

On the new page that opens, select “Custom Digest” under the “Main Options” heading on the left side of the page.

The new page that opens will contain a list of restriction enzymes. Select the enzymes BamHI and EcoRI then click on the “Digest” button at the bottom of the page.

On the new page that opens you will see a linear representation of your DNA. Select “Fragments” under the “List” heading on the right side of the page.

Record the length of the longest fragment in Table 1. This fragment contains the sequence of the foreign DNA. The short fragments are the left over ends from the restriction enzyme digest. Close this window.

Return to the NEB Cutter homepage. Copy and paste the plasmid gene sequence into the box on the NEB Cutter website where it says “Paste In Your DNA Sequence”. Plasmid DNA Sequence:

GTTAACTACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTG

TTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATA

AATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCG

CCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACG

CTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATC

GAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGT

TCTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTG

TTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACT

TGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAG

AGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTT

CTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGG

GATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAA

ACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAAC

TATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATG

GAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGG

TTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGC

ACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGT

CAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGAT

TAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTACCCC

GGTTGATAATCAGAAAAGCCCCAAAAACAGGAAGATTGTATAAGCAAATATTTAAATT

GTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTT

AACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGCCCGAGATA

GGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCC

AACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATC

ACCCAAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAA

AGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCGAACGTGGCGAGAAAGGAAG

GGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGC

TGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTAAAA

GGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTT

TTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCC

TTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGT

GGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAG

CAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTT

CAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGC

TGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACC

GGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTG

GAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGC

CACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGA

ACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCC

TGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGG

GCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTG

CTGGCCTTTTGCTCACATGTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTT

ACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCAC

ACAGGAAACAGCTATGACCATGATTACGCCAAGCTACGTAATACGACTCACTATAGGG

CAGATCTTCGAATGCATCGCGCGCACCGTACGTCTCGAGGAATTCCTGCAGGATATC

TGGATCCACGAAGCTTCCCATGGTGACGTCACCGGTTCTAGATACCTAGGTGAGCTC

TGGTACCCTCTAGTCAAGGCCTATAGTGAGTCGTATTACGGACTGGCCGTCGTTTTAC

AACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCC

CCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACA

GTTGCGCAGCCTGAATGGCGAATGGCGCTTCGCTTGGTAATAAAGCCCGCTTCGGCG

GGCTTTTTTTT

Title this sequence as “Plasmid DNA” in the “Name of Sequence” box. Select “Circular” by the heading “The sequence is:”. Leave the other settings on their default status.

Click the “Submit” button (located to the right of the pasted DNA sequence).

On the new page that opens, select “Custom Digest” under the “Main Options” heading on the left side of the page.

The new page that opens will contain a list of restriction enzymes. Select the enzymes BamHI and EcoRI then click on the “Digest” button at the bottom of the page.

On the new page that opens you will see a circular representation of your DNA. Select “Fragments” under the “List” heading on the right side of the page.

Record the length of the longest fragment in Table 1. This fragment contains the majority of the plasmid DNA sequence. The short fragment is the excised plasmid DNA that lies in between the two restriction enzyme sites.

Table 1: Fragment Lengths
DNA Type Longest Length (in base pairs)
Foreign
Plasmid

Q: Identify where the common restriction sites for both sequences occur?

Solutions

Expert Solution

The final results after completing all the steps are as below:

The restriction site after custom digest is shown below:

As we can see, in foreign DNA, ECoRI site is interfered by CpG island. So only BamHI is present in boh foreign DNA and plasmid DNA.

Identify where the common restriction sites for both sequences occur?

Common restriction sites occur by BamHI


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