Protein
domain
BioBricks
Perry Tsai
June 19, 2006
Goals
 (1)
Demonstrate the use of protein
domains in BioBricks assembly (hDlg
experiment)
 (2) Develop library of BioBrick’d protein
domains (populate the registry)
 (3) Design and construct a novel
protein (for its own sake or for other
project)
http://www.criduchat.asn.au/criduchat/Images/notebook.JPG
The pCMV mammalian expression vector
http://www.stratagene.com/vectors/maps/pdf/pCMV-Tag3A.pdf
The pCMV mammalian expression vector
http://www.stratagene.com/vectors/maps/pdf/pCMV-Tag3A.pdf
The pCMV mammalian expression vector
http://www.stratagene.com/vectors/maps/pdf/pCMV-Tag3A.pdf
The pCMV mammalian expression vector
http://www.stratagene.com/vectors/maps/pdf/pCMV-Tag3A.pdf
The pCMV mammalian expression vector
http://www.stratagene.com/vectors/maps/pdf/pCMV-Tag3A.pdf
The pCMV mammalian expression vector
http://www.stratagene.com/vectors/maps/pdf/pCMV-Tag3A.pdf
Converting multiple cloning site to BioBricks site
pCMV-Tag3A
vector
BamHI/XhoI
digest
BamHI
sticky ends
XhoI
Converting multiple cloning site to BioBricks site
BamHI
BamHI/XhoI
digest
pCMV-Tag3A
vector
sticky ends
XhoI
+
BioBricks linker
5’-GATCCTGAATTCGCGGCCGCTTCTAGAGTACTAGTAGCGGCCGCTGCAGC
-3’
3’GACTTAAGCGCCGGCGAAGATCTCATGATCATCGCCGGCGACGTCGAGCT -5’
BamHI
EcoRI
NotI
XbaI
SpeI
NotI
PstI
XhoI
Converting multiple cloning site to BioBricks site
BamHI
BamHI/XhoI
digest
pCMV-Tag3A
vector
sticky ends
XhoI
+
BioBricks linker
5’-GATCCTGAATTCGCGGCCGCTTCTAGAGTACTAGTAGCGGCCGCTGCAGC
-3’
3’GACTTAAGCGCCGGCGAAGATCTCATGATCATCGCCGGCGACGTCGAGCT -5’
BamHI
EcoRI
NotI
XbaI
SpeI
NotI
ligase
pCMV-Tag3A-BB
vector
BioBricks site
pCMV-Tag3A-BB
PstI
XhoI
Converting protein domains
to BioBricks
EcoRI
NotI
XbaI
5’ GTTTCTCCGAATTCGCGGCCGCTTCTAGAG[Part, 5’->3’ sense]
Protein domain coding sequence
5’
3’
3’
5’
[Part, 3’->5’ antisense]-ATGATCATCGCCGGCGACGTCCCTCTTTG 3’
SpeI
NotI
PstI
Converting protein domains
to BioBricks
EcoRI
NotI
XbaI
5’ GTTTCTCCGAATTCGCGGCCGCTTCTAGAG[Part, 5’->3’ sense]
Protein domain coding sequence
5’
3’
3’
5’
[Part, 3’->5’ antisense]-ATGATCATCGCCGGCGACGTCCCTCTTTG 3’
SpeI
NotI
PstI
PCR
Protein domain coding sequence
5’ GTTTCTCCGAATTCGCGGCCGCTTCTAGAG
3’ CAAAGAGGCTTAAGCGCCGGCGAAGATCTC
EcoRI
NotI
XbaI
TACTAGTAGCGGCCGCTGCAGGGAGAAAC 3’
ATGATCATCGCCGGCGACGTCCCTCTTTG 5’
SpeI
NotI
PstI
Converting protein domains
to BioBricks
EcoRI
NotI
XbaI
5’ GTTTCTCCGAATTCGCGGCCGCTTCTAGAG[Part, 5’->3’ sense]
Protein domain coding sequence
5’
3’
3’
5’
[Part, 3’->5’ antisense]-ATGATCATCGCCGGCGACGTCCCTCTTTG 3’
SpeI
NotI
PstI
PCR
Protein domain coding sequence
5’ GTTTCTCCGAATTCGCGGCCGCTTCTAGAG
3’ CAAAGAGGCTTAAGCGCCGGCGAAGATCTC
EcoRI
NotI
XbaI
TACTAGTAGCGGCCGCTGCAGGGAGAAAC 3’
ATGATCATCGCCGGCGACGTCCCTCTTTG 5’
SpeI
NotI
PstI
PstI
XhoI
TOPO cloning
Protein domain
BioBrick
TOPO vector
Caveat: BioBricks restriction sites need to be removed from the coding sequence via
site-directed mutagenesis.
The hDlg protein
• Human homolog of Drosophila Discs-large tumor
suppressor
• Alternatively spliced isoforms
McLaughlin M, Hale R, Ellston D, Gaudet S, Lue RA, and Viel A. The distribution and function of alternatively spliced insertions in hDlg.
J Biol Chem 2002 Feb 22; 277(8) 6406-12.
The hDlg protein
• Human homolog of Drosophila Discs-large tumor
suppressor
• Alternatively spliced isoforms
• Insertions I2, I3, I4, I5
McLaughlin M, Hale R, Ellston D, Gaudet S, Lue RA, and Viel A. The distribution and function of alternatively spliced insertions in hDlg.
J Biol Chem 2002 Feb 22; 277(8) 6406-12.
The hDlg protein
• Human homolog of Drosophila Discs-large tumor
suppressor
• Alternatively spliced isoforms
• Insertions I2, I3, I4, I5
• I3 targets to plasma membrane; I2 targets to nucleus
McLaughlin M, Hale R, Ellston D, Gaudet S, Lue RA, and Viel A. The distribution and function of alternatively spliced insertions in hDlg.
J Biol Chem 2002 Feb 22; 277(8) 6406-12.
Mix and match
F11
PCR to create BioBrick parts 1-9
SG25
SG35
I2-I5-I4
Mix and match
F11
PCR to create BioBrick parts 1-9
SG25
SG35
I2-I5-I4
Assemble into constructs
Progress and agenda




pCMV-Tag3BB vector and BioBrick parts 1-9 have been created,
sequenced, and midiprepped. Assemblies have been attempted but
not verified.
Next, assemblies need to be completed and verified by sequencing.
(3 wks)
Then, constructs can be transfected into human epithelial cells,
selected by kanamycin resistance.
(2 wks)
Finally, localization of hDlg isoforms will be tested by immunostaining
for myc tag.
(2 wks)
Pros/Cons





Achievable in 1-2 months by 2-3 people
An observable proof of principle
The principle has already been proven. (Silver lab)
Not creative
Not iGEM-y
http://www.physlink.com/estore/cart/item_images/780_xl.jpg
Protein domain
BioBricks library


Develop a BioBricks library of protein domains within the Registry
Mix and match protein domains




Model alternatively spliced proteins, like in hDlg experiment
Add, remove, or replace domains for altered functionality
Fuse domains together into chimeric proteins
Design new proteins from characterized domains
http://parts.mit.edu/registry/index.php/Image:Registry-head-c2g.png
Types of domains






Receptor (iron, aspartate)
Kinase (tyrosine, serine)
Transmembrane (OmpX)
Localization (nuclear, membrane)
Targeting (intercell)
DNA binding (repressor)
http://olenka.med.virginia.edu/mcsg/images/structures/2AU5x500r.jpg
Sources of domains

Genomes (E. coli, human, yeast)
 cDNA, clone libraries
 Synthesis
 Mutation
 Evolution
http://olenka.med.virginia.edu/mcsg/images/structures/2AU5x500r.jpg
Agenda





Find out what domains Silver lab has BioBrick’d and what may be
added to the Registry.
Research other interesting domains, design primers, order primers,
DNA sources, and plasmids.
(2 wks)
BioBrick parts from chosen protein domains, add BioBricks sites to
plasmids if necessary, clone into plasmids.
.
(3 wks)
Modify parts as needed (remove restriction sites, mutations). (1 wk)
Verify parts by sequencing.
(1 wk)
Pros/Cons

Flexible timeline and goals



Estimated goal of 10-15 parts in 2 months by 3-4 people
Independent chances at success and failure
Potential to plan for more parts if ahead of schedule

Important for future iGEM teams, future protein domain research
 Silver lab experience

Not terribly exciting
 Not creative, except choice/modification of parts
http://www.physlink.com/estore/cart/item_images/780_xl.jpg
Designing/constructing
a novel protein
 Use
BioBrick’d protein domains to build
a new protein
 Can be any desired protein, or a protein
useful for another project
http://www.cygnus-x1.net/links/lcars/blueprints/star-trek-the-motion-picture-sheet-4.jpg
Chimeric protein examples

LamB secretion signal + mature bovine somatotropin


Aspartate chemoreceptor + EnvZ activator of OmpC transcription


Moe GR, Bollag GE, and Koshland DE Jr. Transmembrane signaling by a chimera of the Escherichia coli aspartate receptor and the human insulin receptor. Proc
Natl Acad Sci U S A 1989 Aug; 86(15) 5683-7.
Iron receptor + beta-galactosidase


Utsumi R, Brissette RE, Rampersaud A, Forst SA, Oosawa K, and Inouye M. Activation of bacterial porin gene expression by a chimeric signal transducer in
response to aspartate. Science 1989 Sep 15; 245(4923) 1246-9.
Aspartate chemoreceptor + human insulin receptor kinase


Klein BK, Hill SR, Devine CS, Rowold E, Smith CE, Galosy S, and Olins PO. Secretion of active bovine somatotropin in Escherichia coli. Biotechnology (N Y)
1991 Sep; 9(9) 869-72.
Coulton JW, Mason P, Cameron DR, Carmel G, Jean R, and Rode HN. Protein fusions of beta-galactosidase to the ferrichrome-iron receptor of Escherichia coli
K-12. J Bacteriol 1986 Jan; 165(1) 181-92.
Maltose-binding protein + SH2 tyrosine kinase substrate

Sierke SL and Koland JG. SH2 domain proteins as high-affinity receptor tyrosine kinase substrates.
http://www.planetadnd.com/interactive_books/chimera.gif
Agenda






Design a novel protein, and research necessary domains. Design
primers. Order DNA source, primer, plasmid.
(1 wk)
BioBrick domains, add BioBrick site to plasmid if necessary, clone
into plasmid.
(2 wks)
Modify parts as needed (remove restriction sites)
(1 wk)
Verify parts by sequencing, while beginning assembly.
(1 wk)
Complete assembly, and verify by sequencing.
(2 wks)
Test functionality of construct.
(1 wk)
Pros/Cons





Creative exercise
Applicable and useful to many potential projects
Difficult to ensure success with a novel protein
Protein domains may not be so modular
Tighter timeline because of BioBricking + assembly.
http://www.physlink.com/estore/cart/item_images/780_xl.jpg
Final thoughts






3 projects: (1) hDlg, (2) library, (3) novel protein
Interestingness? 1 < 2 < 3
1 is “easily” achievable
2 & 3 have overlapping agendas; feasible with 4-5
people working over 2 months.
2 & 3 require further research
1 & 2 are independent projects, while 3 may
depend on 2, and another project may depend on 3