GENETIC TRANSFORMATION:
SEQUENCE EXPLORATION
You will use a DNA search engine called Nucleotide BLAST at the National Center for
Biotechnology Information (NCBI) website to search GenBank, a fully annotated
database of all publicly available DNA sequences and their protein translations, for the
foreign gene used in the Genetic Transformation Lab. Sequences in GenBank are
contributed by individual labs and sequencing facilities all over the world. As of April
2008, there were more than 76 million individual sequences from over 260,000
organisms in the GenBank database.
PART I: BLAST N SEARCH
Below is the wild-type (natural, unaltered) DNA sequence of the foreign gene which we
have been exploring in our Genetic Transformation Lab. This is GeneG that is contained
in the pYSPG plasmid that some of you transformed into bacteria. This gene is 717
nucleotide bases long, including the start codon (ATG) and the stop codon (TAA). Here
is the sequence of GeneG:
>>GeneG
1 ATGAGTAAAG GAGAAGAACT TTTCACTGGA GTGGTCCCAG TTCTTGTTGA
ATTAGATGGC
61 GATGTTAATG GGCAAAAATT CTCTGTCAGT GGAGAGGGTG AAGGTGATGC AACATACGGA
121 AAACTTACCC TTAATTTTAT TTGCACTACT GGGAAGCTAC CTGTTCCATG GCCAACACTT
181 GTCACTACTT TCTCTTATGG TGTTCAATGC TTCTCAAGAT ACCCAGATCA TATGAAACAG
241 CATGACTTTT TCAAGAGTGC CATGCCCGAA GGTTATGTAC AGGAAAGAAC TATATTTTAC
301 AAAGATGACG GGAACTACAA GACACGTGCT GAAGTCAAGT TTGAAGGTGA TACCCTTGTT
361 AATAGAATCG AGTTAAAAGG TATTGATTTT AAAGAAGATG GAAACATTCT TGGACACAAA
421 ATGGAATACA ACTATAACTC ACATAATGTA TACATCATGG GAGACAAACC AAAGAATGGC
481 ATCAAAGTTA ACTTCAAAAT TAGACACAAC ATTAAAGATG GAAGCGTTCA ATTAGCAGAC
541 CATTATCAAC AAAATACTCC AATTGGCGAT GGCCCTGTCC TTTTACCAGA CAACCATTAC
601 CTGTCCACAC AATCTGCCCT TTCCAAAGAT CCCAACGAAA AGAGAGATCA CATGATCCTT
661 CTTGAGTTTG TAACAGCTGC TAGGATTACA CATGGCATGG ATGAACTATA CAAATAA
1. To access the NCBI Nucleotide BLAST webpage, right-click on the following link
and select ‘Open Hyperlink’:
http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&BLAST_PROGRAMS=
megaBlast&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=bla
sthome
2. Where it says ‘Enter Query Sequence’, copy and paste the GeneG sequence
(including the >>GeneG header) into the box provided. It does not matter if there are
numbers in the sequence. (This is illustrated on the following page.)
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3. Where it says ‘Choose Search Set’, select ‘Nucleotide collection (nr/nt)’ from the
drop-down Database menu.
4. Leave all other fields blank.
5. Click on the
button at the bottom left side of the page.
6. Wait a few moments while the program searches GenBank for a match to the foreign
gene.
7. You should now have a page with your search results. Scroll-down to the
‘Descriptions’ section and look at the results. Notice that they are ranked by scores,
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from highest to lowest. Score indicates the degree of identity between your query
sequence and the sequence in the database. Accession Number is the identifier that
has been assigned to a particular sequence in the database.
8. Click on the ‘Accession Number’ link for the top hit, i.e. the one with the highest
score which should be at the top of the list. This will take you to the GenBank
information page for that sequence.
What protein is encoded by GeneG?
What organism is this gene from?
PART II: DNA SEQUENCE ALIGNMENT
The pYSPB plasmid that some of you transformed into bacteria contains a mutated
version of the foreign gene, designated GeneB. Below is the sequence of GeneB:
>>GeneB
1
61
121
181
241
301
361
421
481
541
601
661
ATGAGTAAAG
GATGTTAATG
AAACTTACCC
GTCACTACTT
CATGACTTTT
AAAGATGACG
AATAGAATCG
ATGGAATACA
ATCAAAGTTA
CATTATCAAC
CTGTCCACAC
CTTGAGTTTG
GAGAAGAACT
GGCAAAAATT
TTAATTTTAT
TCTCTCACGG
TCAAGAGTGC
GGAACTACAA
AGTTAAAAGG
ACTATAACTC
ACTTCAAAAT
AAAATACTCC
AATCTGCCCT
TAACAGCTGC
TTTCACTGGA
CTCTGTCAGT
TTGCACTACT
TGTTCAATGC
CATGCCCGAA
GACACGTGCT
TATTGATTTT
ACATAATGTA
TAGACACAAC
AATTGGCGAT
TTCCAAAGAT
TAGGATTACA
GTGGTCCCAG
GGAGAGGGTG
GGGAAGCTAC
TTCTCAAGAT
GGTTATGTAC
GAAGTCAAGT
AAAGAAGATG
TACATCATGG
ATTAAAGATG
GGCCCTGTCC
CCCAACGAAA
CATGGCATGG
TTCTTGTTGA
AAGGTGATGC
CTGTTCCATG
ACCCAGATCA
AGGAAAGAAC
TTGAAGGTGA
GAAACATTCT
GAGACAAACC
GAAGCGTTCA
TTTTACCAGA
AGAGAGATCA
ATGAACTATA
ATTAGATGGC
AACATACGGA
GCCAACACTT
TATGAAACAG
TATATTTTAC
TACCCTTGTT
TGGACACAAA
AAAGAATGGC
ATTAGCAGAC
CAACCATTAC
CATGATCCTT
CAAATAA
You will use an online program to compare the sequences of the wild-type (GeneG) and
mutated (GeneB) genes; this is known as a DNA sequence alignment. An alignment uses
an algorithm (a step-by-step procedure) to compare the order of nucleotide bases in the
sequences and then lines them up so that the number of identical bases is maximized.
The alignment program will point out those bases that are identical (indicated by an
asterisk - ), those that are similar (:), and those that are completely different (no
symbol). Alignments are useful to study how closely genes are related which then allows
evolutionary relationships to be determined. For example, how are genes that code for
the same protein in different species or genes passed on between generations related?
You will use the ClustalW2 general sequence alignment tool provided by the European
Bioinformatics Institute.
1. Right-click on the following link and select ‘Open Hyperlink’ to access the
ClustalW2 website: http://www.ebi.ac.uk/Tools/clustalw2/index.html
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2. Where it says “Enter your input sequences”, select DNA in the drop down menu
and paste the GeneB and GeneG sequences.
a. First copy and paste the GeneG sequence (including the >>GeneG header)
into the box.
b. Press Enter to leave a space after the GeneG sequence.
c. Then copy and paste the GeneB sequence (including the >>GeneB header)
below the GeneG sequence in the same box.
3. Change the input sequence to DNA from Protein, in the Step 1 window. There is no
need to change any of the other default parameters.
4. Click on
5. You may have to wait a few moments while the alignment program is running.
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6.
Once it loads, copy and paste the alignment results into the box provided below.
Can you identify which nucleotide bases have been mutated in GeneB?
Alignment of GeneG and GeneB Nucleotide Sequences:
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PART III: DNA TRANSLATION AND PROTEIN SEQUENCE ALIGNMENT
The ClustalW2 program can also be used to align protein (amino acid) sequences. First,
you will use a tool provided by Colorado State University to translate the sequences of
GeneG and GeneB into their corresponding amino acid sequences. Then, you will use
the ClustalW2 program to align the amino acid sequences and identify which amino
acid(s) is mutated in GeneB.
1. Right-click on the following link and select ‘Open Hyperlink’ to access the website:
http://www.vivo.colostate.edu/molkit/translate/
2. In the first empty box, copy and paste the sequence for GeneG – this time, DO NOT
include the >>GeneG header (but numbers can be included).
3. Click on ‘Translate DNA’. (Do not change any of the other options.)
4. Click on ‘Text Output’.
5. You should see the amino acid translation above the DNA sequence. Amino acids
are given in one-letter code. Recall that three nucleotide bases code for one amino
acid.
6. In the third drop-down menu from the left it currently says ‘Amino acids and DNA’,
select ‘Amino acids only’.
7. You should now see only the amino acid sequence translated from the gene.
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8. Copy and paste the amino acid translation for GeneG (from the number 1 to the end
of the sequence) in the space provided below.
9. Clear the DNA by pressing the Clear DNA button.
.
DO NOT select the text and delete it, YOU MUST press the Clear DNA button.
10. Repeat steps 2 to 8 for GeneB.
>>GeneG_aminoacid
>>GeneB_aminoacid
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11. Go back to the ClustalW2 website (see pg.4 instructions). This time, copy and paste
the amino acid sequences translated from GeneG and GeneB into the query box (one
below the other, including the >> headers).
12. Click on ‘Run’. And paste the sequence below:
13. Below, you are provided with the one- and three-letter abbreviations for the 20
common amino acids. Examine the alignment results. Can you identify what
amino acid mutation has occurred in GeneB?
One letter code
A
C
D
E
F
G
H
I
K
L
M
N
P
Q
R
S
T
V
W
Y
Three letter code
ala
cys
asp
glu
phe
gly
his
ile
lys
leu
met
asn
pro
gln
arg
ser
thr
val
try
tyr
Amino acid
alanine
cysteine
aspartic acid
glutamic acid
phenylalanine
glycine
histidine
isoleucine
lysine
leucine
methionine
asparagine
proline
glutamine
arginine
serine
threonine
valine
tryptophan
tyrosine
14. Can you manually transcribe a DNA sequence into its complementary RNA and
translate that RNA into its corresponding amino acid sequence? Of course!
Scientists have uncovered the Universal Genetic Code which describes how a specific
three nucleotide codon translates for a particular amino acid. Right-click on the
following link and select ‘Open Hyperlink’:
http://learn.genetics.utah.edu/content/begin/dna/transcribe/
15. After you have gone through the exercise, go back to your nucleotide sequence
alignment results (on pg. 5).
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Can you identify the codon responsible for the amino acid mutation seen in
GeneB?
When you go back to the lab later today, you will discover what effect this mutation
has on the phenotype of the E.coli cells which have been transformed with pYSPB
compared to pYSPG!
PART IV: DISCOVER MORE!
Use your favourite search engine (e.g. Google) to discover more about the foreign
gene (GeneG) and the protein it encodes! What are some special properties of this
protein?
Who won the Nobel Prize for its discovery and development?
What are some of the ways scientists have used this gene for scientific research?
WANT TO LEARN MORE ABOUT DNA SCIENCE?!
DNA Today! To find out more about the role of DNA science in your lives, join
commentators Dave Micklos and Jan Witkowski from world-renowned Cold Spring
Harbor Laboratory for a lively discussion of DNA in the news:
http://www.dnalc.org/ddnalc/dna_today/index.html
DNA from the Beginning! Visit http://www.dnaftb.org/ to discover the concepts and
experiments that define the fields of genetics and molecular biology. This animated
primer features the work of over 100 scientists and researchers.
DNA Timeline! Travel through time with scientists from the first discovery of DNA to
sequencing of the human genome at http://www.dnai.org/timeline/index.html.

To learn more about GenBank, visit:
http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=18073190
Note: For the purposes of these exercises the wild-type gene sequence of GFP was provided as GeneG, in actuality the gene
transformed into bacteria in the laboratory experiment was EGFP (Enhanced GFP) which contains several amino acid substitutions
which allow the fluorescence of the protein to be brighter and last longer. The sequence for GeneB given in this exercise was
constructed based on the knowledge that a single amino acid point mutation (Y66H) is responsible for the blue fluorescence seen in
the GFP variant known as BFP (Heim et al., PNAS USA, 91:12501-12504, 1994); in actuality the gene transformed into bacteria in the
laboratory exercise was EBFP (Enhanced BFP) which in addition to the Y66H mutation, contains several other amino acid mutations
which similarly allow the blue fluorescence to shine brighter and last longer.
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