Assignment Project in Chemical Biology, TFKE39
You have been given the task of isolating any of these 10 DNA sequences coding for a
protein. The gene isolated from a cDNA library and cloned into an expression vector for
expressing the protein in E. coli.
Your task will be to make a laboratory protocols in which you describe:
Which protein does the DNA sequence codes for?
Indicate how you came to this conclusion, which database did you use?
Designing primers for amplification of the gene with the help of PCR
Designing primers to amplify the gene based on a cDNA library, note 5 'or 3' end and the
forward and reverse primers. Specify how it binds to DNA (eg mark with italics or bold)
Justify your choice of primer construction and how you chose the PCR cycle to amplify
the gene.
Isolation of PCR fragments
Specify how you purify the PCR fragment after amplification.
Choice of vectors for expression in E. coli
Justify your choice of vector, based on the information you received about the protein's
properties (eg, large or small protein?, Membrane protein or cytosolic? Post
translationally modified?)
Strategy for cloning the gene into the selected vector
How should the gene be cloned into the selected vector? Which restriction enzymes
should be used? How should the gene be expressed? Under what conditions should it be
done (culture temperature, antibiotic resistance, induction, with what?
The chance that the protein is able to express in E. coli
Based on the properties of the protein (eg, large or small protein?, Membrane protein or
cytosolic? Post translational modified?) Speculate on the chance that your design will be
possible to express in E. coli
Describe methods how to characterize the protein
Based on the protein function make suggestions about how to characterize the protein (eg,
the protein enzyme activity? Is it possible to measure the protein stability with a
spectroscopic method?
The assignment will be sent to [email protected] at latest Wednesday 26th of october.
NOTE: Submitted report should be in PDF format
Late assignments without justification will not be read.
Lasse Martensson
IFM-Chemistry
[email protected]
Assessment: Individual Project Plan 5-10 pages that are considered according to the
attached evaluation grid with scale (U, 3,4,5)
References
Literature:
• TA Brown, Gene Cloning and DNA Analysis, An Introduction
Databases and molecular biology companies:
• Expasy Proteomics Server
http://www.expasy.ch/
Protein Data Bank (3d structures of proteins)
http://www.rcsb.org
• NEB Cutter On-line program to check the restriction sites
http://tools.neb.com/NEBcutter2/index.php
Companies that sell molecular biology products or services
• Stratagene (including the QuikChange site-specific mutagenesis)
http://www.stratagene.com
• New England Biolabs (NEB) (among others restriction enzymes)
http://www.neb.com
• Qiagen (including products for the purification of DNA, plasmidprep kit)
http://www.qiagen.com
• Novagen (including vectors for expression of proteins)
http://www.novagen.com
• GATC (including performing DNA sequencing
http://www.gatc.de
Choose one (1) of the following DNA sequence for cloning and expression in E.coli
A
atgagtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggt
gatgttaatgggcacaaattctctgtcagtggagagggtgaaggtgatgcaacatacgga
aaacttacccttaaatttatttgcactactggaaagctacctgttccatggccaacactt
gtcactactttctcttatggtgttcaatgcttttcaagatacccagatcatatgaaacag
catgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatattttac
aaagatgacgggaactacaaatcacgtgctgaagtcaagtttgaaggtgataccctcgtt
aatagaattgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaa
atggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatgga
atcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagac
cattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattac
ctgtccacacaatctgccctttccaaagatcccaacgaaaagagagatcacatgatcctt
cttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaataa
B
atggcttctcatcgtctgctcctcctctgccttgctggactggtatttgtgtctgaggct
ggccctacgggcaccggtgaatccaagtgtcctctgatggtcaaagttctagatgctgtc
cgaggcagtcctgccatcaatgtggccgtgcatgtgttcagaaaggctgctgatgacacc
tgggagccatttgcctctgggaaaaccagtgagtctggagagctgcatgggctcacaact
gaggaggaatttgtagaagggatatacaaagtggaaatagacaccaaatcttactggaag
gcacttggcatctccccattccatgagcatgcagaggtggtattcacagccaacgactcc
ggcccccgccgctacaccattgccgccctgctgagcccctactcctattccaccacggct
gtcgtcaccaatcccaaggaatga
C
atgaactaccccgtgaatcccgacctgatgcccgcgctgatggcggtcttccagcatgtg
cggacgcgcatccagagcgagctcgattgccagcgactcgacctgaccccgcccgacgtc
catgtattgaagcttatcgacgaacaacgcgggctgaacctgcaggacctgggacgccag
atgtgccgcgacaaggcactgatcacccggaagatccgcgagctggagggaagaaacctg
gtccgccgcgagcgcaaccccagcgaccagcgcagcttccagctcttcctcaccgacgag
gggctggccatccaccagcatgcggaggccatcatgtcacgcgtgcatgacgagttgttt
gccccgctcaccccggtggaacaggccaccctggtgcatctcctcgaccagtgcctggcc
gcgcaaccgcttgaggatatttaa
D
atgccccggcgcagcctgcacgcggcggccgtgctcctgctggtgatcttaaaggaacag
ccttccagcccggccccagtgaacggttccaagtggacttattttggtcctgatggggag
aatagctggtccaagaagtacccgtcgtgtgggggcctgctgcagtcccccatagacctg
cacagtgacatcctccagtatgacgccagcctcacgcccctcgagttccaaggctacaat
ctgtctgccaacaagcagtttctcctgaccaacaatggccattcagtgaagctgaacctg
ccctcggacatgcacatccagggcctccagtctcgctacagtgccacgcagctgcacctg
cactgggggaacccgaatgacccgcacggctctgagcacaccgtcagcggacagcacttc
gccgccgagctgcacattgtccattataactcagacctttatcctgacgccagcactgcc
agcaacaagtcagaaggcctcgctgtcctggctgttctcattgagatgggctccttcaat
ccgtcctatgacaagatcttcagtcaccttcaacatgtaaagtacaaaggccaggaagca
ttcgtcccgggattcaacattgaagagctgcttccggagaggaccgctgaatattaccgc
taccgggggtccctgaccacacccccttgcaaccccactgtgctctggacagttttccga
aaccccgtgcaaatttcccaggagcagctgctggctttggagacagccctgtactgcaca
cacatggacgacccttcccccagagaaatgatcaacaacttccggcaggtccagaagttc
gatgagaggctggtatacacctccttctcccaagtgcaagtctgtactgcggcaggactg
agtctgggcatcatcctctcactggccctggctggcattcttggcatctgtattgtggtg
gtggtgtccatttggcttttcagaaggaagagtatcaaaaaaggtgataacaagggagtc
atttacaagccagccaccaagatggagactgaggcccacgcttga
E
atggatggtacaagaacttcacttgacattgaagagtactcggatactgaggtacagaaa
aaccaagtactaactctggaagaatggcaagacaagtgggtgaacggcaagactgctttt
catcaggaacaaggacatcagctattaaagaagcatttagatactttccttaaaggcaag
agtggactgagggtattttttcctctttgcggaaaagcggttgagatgaaatggtttgca
gaccggggacacagtgtagttggtgtggaaatcagtgaacttgggatacaagaatttttt
acagagcagaatctttcttactcagaagaaccaatcaccgaaattcctggaaccaaagta
tttaagagttcttcggggaacatttcattgtactgttgcagtatttttgatcttcccagg
acaaatattggcaaatttgacatgatttgggatagaggagcattagttgccattaatcca
ggtgatcgcaaatgctatgcagatacaatgttttccctcctgggaaagaagtttcagtat
ctcctgtgtgttctttcttatgatccaactaaacatccaggtccaccattttatgttcca
catgctgaaattgaaaggttgtttggtaaaatatgcaatatacgttgtcttgagaaggtt
gatgcttttgaagaacgacataaaagttggggaattgactgtctttttgaaaagttatat
ctacttacagaaaagtaa
F
atgagctcccagattcgtcagaattattccaccgacgtggaggcagccgtcaacagcctg
gtcaatttgtacctgcaggcctcctacacctacctctctctgggcttctatttcgaccgc
gatgatgtggctctggaaggcgtgagccacttcttccgcgaactggccgaggagaagcgc
gagggctacgagcgtctcctgaagatgcaaaaccagcgtggcggccgcgctctcttccag
gacatcaagaagccagctgaagatgagtggggtaaaaccccagacgccatgaaagctgcc
atggccctggagaaaaagctgaaccaggcccttttggatcttcatgccctgggttctgcc
cgcacggacccccatctctgtgacttcctggagactcacttcctagatgaggaagtgaag
cttatcaagaagatgggtgaccacctgaccaacctccacaggctgggtggcccggaggct
gggctgggcgagtatctcttcgaaaggctcactctcaagcacgactaa
G
atgacagcagacaaattggttttctttgtgaatggcagaaaggtggtggagaaaaatgca
gatccagagacaacccttttggcctacctgagaagaaagttggggctgagtggaaccaag
ctcggctgtggagaggggggctgcggggcttgcacagtgatgctctccaagtatgatcgt
ctgcagaacaagatcgtccacttttctgccaatgcctgcctggcccccatctgctccttg
caccatgttgcagtgacaactgtggaaggaataggaagcaccaagacgaggctgcatcct
gtgcaggagagaattgccaaaagccacggctcccagtgcgggttctgcacccctggcatc
gtcatgagtatgtacacactgctccggaatcagcccgagcccaccatggaggagattgag
aatgccttccaaggaaatctgtgccgctgcacaggctacagacccatcctccagggcttc
cggacctttgccagggatggtggatgctgtggaggagatgggaataatccaaattgctgc
atgaaccagaagaaagaccactcagtcagcctctcgccatctttattcaaaccagaggag
ttcacgcccctggatccaacccaggagcccatttttcccccagagttgctgaggctgaaa
gacactcctcggaagcagctgcgatttgaaggggagcgtgtgacgtggatacaggcctca
accctcaaggagctgctggacctcaaggctcagcaccctgacgccaagctggtcgtgggg
aacacggagattggcattgagatgaagttcaagaatatgctgtttcctatgattgtctgc
ccagcctggatccctgagctgaattcggtagaacatggacccgacggtatctcctttgga
gctgcttgccccctgagcattgtggaaaaaaccctggtggatgctgttgctaagcttcct
gcccaaaagacagaggtgttcagaggggtcctggagcagctgcgctggtttgctgggaag
caagtcaagtctgtggcgtccgttggagggaacatcatcactgccagccccatctccgac
ctcaaccccgtgttcatggccagtggggccaagctgacacttgtgtccagaggcaccagg
agaactgtccagatggaccacaccttcttccctggctacagaaagaccctgctgagcccg
gaggagatactgctctccatagagatcccctacagcagggagggggagtatttctcagca
ttcaagcaggcctcccggagagaagatgacattgccaaggtaaccagtggcatgagagtt
ttattcaagccaggaaccacagaggtacaggagctggccctttgctatggtggaatggcc
aacagaaccatctcagccctcaagaccactcagaggcagctttccaagctctggaaggag
gagctgctgcaggacgtgtgtgcaggactggcagaggagctgcatctgcctcccgatgcc
cctggtggcatggtggacttccggtgcaccctcaccctcagcttcttcttcaagttctac
ctgacagtccttcagaagctgggccaagagaacctggaagacaagtgtggtaaactggac
cccactttcgccagtgcaactttactgtttcagaaagaccccccagccgatgtccagctc
ttccaagaggtgcccaagggtcagtctgaggaggacatggtgggccggcccctgccccac
ctggcagcggacatgcaggcctctggtgaggccgtgtactgtgacgacattcctcgctac
gagaatgagctgtctctccggctggtcaccagcacccgggcccacgccaagatcaagtcc
atagatacatcagaagctaagaaggttccagggtttgtttgtttcatttccgctgatgat
gttcctgggagtaacataactggaatttgtaatgatgagacagtctttgcgaaggataag
gttacttgtgttgggcatatcattggtgctgtggttgctgacaccccggaacacacacag
agagctgcccaaggggtgaaaatcacctatgaagaactaccagccattatcacaattgag
gatgctataaagaacaactccttttatggacctgagctgaagatcgagaaaggggaccta
aagaaggggttttccgaagcagataatgttgtgtcaggggagatatacatcggtggccaa
gagcacttctacctggagactcactgcaccattgctgttccaaaaggcgaggcaggggag
atggagctctttgtgtctacacagaacaccatgaagacccagagctttgttgcaaaaatg
ttgggggttccagcaaaccggattgtggttcgagtgaagagaatgggaggaggctttgga
ggcaaggagacccggagcactgtggtgtccacggcagtggccctggctgcatataagacc
ggccgccctgtgcgatgcatgctggaccgtgatgaggacatgctgataactggtggcaga
catcccttcctggccagatacaaggttggcttcatgaagactgggacagttgtggctctt
gaggtggaccacttcagcaatgtggggaacacccaggatctctctcagagtattatggaa
cgagctttattccacatggacaactgctataaaatccccaacatccggggcactgggcgg
ctgtgcaaaaccaaccttccctccaacacggccttccggggctttggggggccccagggg
atgctcattgccgagtgctggatgagtgaagttgcagtgacctgtgggatgcctgcagag
gaggtgcggagaaaaaacctgtacaaagaaggggacctgacacacttcaaccagaagctt
gagggtttcaccttgcccagatgctgggaagaatgcctagcaagctctcagtatcatgct
cggaagagtgaggttgacaagttcaacaaggagaattgttggaaaaagagaggattgtgc
ataattcccaccaagtttggaataagcttcacagttccttttctgaatcaggcaggagcc
ctacttcatgtgtacacagatggctctgtgctgctgacccacggggggactgagatgggc
caaggccttcataccaaaatggtccaggtggccagtagagctctgaaaatccccacctct
aagatttatatcagcgagacaagcactaacactgtgcccaacacctctcccacggctgcc
tctgtcagcgctgacctcaatggacaggccgtctatgcggcttgtcagaccatcttgaaa
aggctggaaccctacaagaagaagaatcccagtggctcctgggaagactgggtcacagct
gcctacatggacacagtgagcttgtctgccactgggttttatagaacacccaatctgggc
tacagctttgagactaactcagggaaccccttccactacttcagctatggggtggcttgc
tctgaagtagaaatcgactgcctaacaggagatcataagaacctccgcacagatattgtc
atggatgttggctccagtctaaaccctgccattgatattggacaggtggaaggggcattt
gtccagggccttggcctcttcaccctagaggagctacactattcccccgaggggagcctg
cacacccgtggccctagcacctacaagatcccggcatttggcagcatccccattgagttc
agggtgtccctgctccgcgactgccccaacaagaaggccatctatgcatcgaaggctgtt
ggagagccgcccctcttcctggctgcttctatcttctttgccatcaaagatgccatccgt
gcagctcgagctcagcacacaggtaataacgtgaaggaactcttccggctagacagccct
gccaccccggagaagatccgcaatgcctgcgtggacaagttcaccaccctgtgtgtcact
ggtgtcccagaaaactgcaaaccctggtctgtgagggtctaa
H
atggtgaaggtcggtgtcaacggatttggccgtattggccgcctggtcaccagggctgcc
ttctcttgtgacaaagtggacattgttgccatcaacgaccccttcattgacctcaactac
atggtctacatgttccagtatgattctacccacggcaagttcaatggcacagtcaaggct
gagaatgggaagctggtcatcaatgggaaacccatcaccatcttccaggagcgcgatccc
gctaacatcaaatggggtgatgctggtgctgagtatgtcgtggagtctactggcgtcttc
accaccatggagaaggctggggctcacctgaagggtggtgccaaaagggtcatcatctcc
gccccttccgctgatgcccccatgtttgtgatgggtgtgaaccacgagaaatatgacaac
tccctcaagattgtcagcaatgcatcctgcaccaccaactgcttagcccccctggccaag
gtcatccatgacaactttggcatcgtggaagggctcatgaccacagtccatgccatcact
gccactcagaagactgtggatggcccctctggaaagctgtggcgtgatggccgtggggca
gcccagaacatcatccctgcatccactggtgctgccaaggctgtgggcaaggtcatccca
gagctgaacgggaagctcactggcatggccttccgtgttcctacccccaatgtatccgtt
gtggatctgacatgccgcctggagaaacctgccaagtatgatgacatcaagaaggtggtg
aagcaggcggccgagggcccactaaagggcatcctgggctacactgaggaccaggttgtc
tcctgtgacttcaacagcaactcccattcctccacctttgatgctggggctggcattgct
ctcaatgacaactttgtgaagctcatttcctggtatgacaatgaatatggctacagcaac
agggtggtggacctcatggcctacatggcctccaaggagtaa
I
atgttccgtttgccagtttcgcttgctcgctcctccattagccgccagttggccatgcgc
ggctatgccaaggatgtgcgtttcggacccgaggtgcgcgccatgatgctccagggcgtc
gatgtgctggccgatgctgtggcagtgaccatgggccccaagggtcgcaatgtgatcatt
gagcagtcgtggggctcgcccaaaatcaccaaggacggcgtcactgtggccaagtccatt
gagctgaaggacaagttccagaacatcggtgccaagctggtccaggatgtggccaacaat
accaacgaggaggccggtgatggcaccaccacggccaccgttctggctcgcgccattgcc
aaggagggcttcgagaagatctccaagggcgccaatcccgtcgagattcgtcgcggtgtc
atgctggccgttgagaccgtcaaggacaaccttaagaccatgtcgcgccccgttagcacg
cccgaggagatcgcccaggtggccaccatctcggccaacggtgatcaggccatcggcaat
ctcatcagcgaggccatgaagaaggtgggacgcgatggcgttatcaccgtcaaggatggc
aagaccctcaccgatgagttggaggttatcgagggcatgaagttcgatcgcggctacatc
tcgccgtacttcatcaactcctcgaagggcgccaaggtggagttccaagatgccctcctc
ctgctgtccgagaagaagatctcatcggtgcagagcatcattcccgctctggagctggcc
aagtcgcagcgcaagccgctggtcatcattgccgaggatatcgatggtgaggccctgagc
accctggtggtgaaccgcctcaagatcggccttcaggtggctgccgtcaaggcacccggc
ttcggcgacaaccgcaagagcaccctcaccgatatggccatcgcctccggcggcattgtc
ttcggcgatgatgctgatctcgtcaagctggaggacgtcaaggttagcgatttgggccag
gtcggcgaggtggtgatcaccaaggacgacaccctgctgctgaagggcaagggcaagaag
gacgatgtgctccgtcgcgccaatcagataaggaccaagatcgaggacaccacctccgag
tacgagaaggagaagctgcaggagcgtctggcccgcttggcctccggcgttgccctgctg
cgcgttggcggctccagcgaggtggaggtcaacgagaaggaggatcgcgtccacgatgcc
ctgaacgccacccgtgctgccgtcgaagagggaatcgttcccggaggcggcacccgtctc
gtccggttgattgagaagctcgagggtgtcgagaccaccaacgaggatcagaaactcggc
gtggagattgtgcgtcgtgcctcgcgaatgccatgcatgacgattgccaagaacgccggc
gttgatggcgccatggtggtggccaaggtggagaatcaggccggtgactacggctacgat
gcgctgaagggcgaatacggcaacctgatcgagaagggcatcatcgatcccaccaaggtg
gtgcgcaccgccatcacagacgcctccggtgtggcctcactgctgaccaccgccgaggct
gtcgtcaccgagatccccaaggaggatggcgctcctgccatgcccggcatgggtggtatg
ggcggcatgggaggcatgggcggcatgggtggcatgatgtaa
J
atggctatgataccttcaatttcaaagttgctttttgttgcaatatgtctttttgtttat
atgggtttgtcatttggtgatttttctattgtgggttattctcaaaatgacttgacatcc
actgaaagacttattcagctatttgaatcgtggatgttgaagcacaataagatttataag
aatattgatgaaaaaatctacagatttgaaatttttaaagataatcttaaatatattgat
gagacaaataagaaaaataacagttattggcttggattaaatgtgtttgctgatatgagc
aatgatgaattcaaagaaaagtatactggttctattgctggaaattatacaacaaccgaa
ctatcatacgaagaagtgcttaatgatggtgatgtaaatatcccggagtatgtcgattgg
agacaaaaaggagctgtcactcctgtaaaaaatcagggttcttgtggtagttgttgggca
ttctcagctgttgtaactatagagggaataattaagattagaactgggaacttaaatgaa
tactcagagcaagaactgcttgactgcgacagacgtagctacgggtgcaatggaggttac
ccttggagtgcacttcaattagtggctcaatatggtattcactacagaaatacttaccca
tatgagggagtgcaacgttattgtcgctcaagggagaaaggtccttatgcagccaaaacc
gatggggttcgacaagtgcaaccatataatgaaggggctctcttatattcaattgcaaac
caacctgtgagcgttgtccttgaagctgctggaaaagatttccaattatatagaggggga
atatttgtggggccatgcggaaacaaagtagatcatgcagtcgcagcagttgggtatgga
ccaaattacatactcataaagaattcatggggtacaggatggggtgaaaatggatatata
agaatcaaaagaggcactggaaactcctatggagtatgtggactttatacaagctcattc
tatcctgttaaaaactga
Evaluation template for assignment in the project course in Chemical
Biology
Assignment
Project Chemical
Biology Student:
Student:
Basic criteria
1
2
Purpose and
content criteria
Average score:
Criteria includes
(Max:28 points)
2
3
4
1
2
3
4
The assignment linguistically and graphically
consistent.
Adequate factually deep, moderately technical
language
Criteria includes
3
Clear and well-motivated Specific Objectives
4
Adequate background summary
5
Sophisticated methodological content in terms of
both experimental and practical aspects
6
Feasibility - overall assessment based on the
application
7
originality, innovation, creativity!
Feedback:
1
