Technical Specification:A Biosynthetic Approach to
Replacing Scarred post-Infarct Tissue With Healthy Cardiac Tissue
20.020
April 9, 2008
Anna ShcherbinaDerek Ju
Prarthna DesaiAmber Lin
Aditya KohliRobbie Barbero
Michael Oh
Impact of the Project
• Almost 100 percent of living organisms (humans included) have scars of some sort (topical, internal, etc.)
•Topical scars are a commonplace imperfection with a variety of already-existing treatment options
•Of all scars, scarring in the heart is one of the most perilous
•Heart scar tissue forms after heart attacks
•The formation of heart scars after heart attacks results in inefficiency of blood pumping and higher occurrences of arrhythmias
Impact (continued)
• What if we could find a way to actually eliminate the precarious tissue build-up that forms after heart attacks and re-form healthy tissue?
•Our project would propose a new system of dealing with formed scars
•Being able to give back someone his heart tissue could, both literally and figuratively, give back his life
The General Idea:
•Monoclonal antibodies are injected into the bloodstream. •One end binds to cardiac scar tissue.•The other end binds to a SMAD receptor on the engineered bacteria.
•Engineered bacteria are injected into the bloodstream and bind to the monoclonal antibody on the cardiac tissue.
•The bacteria secrete collagenase to digest scar tissue.
The General Idea (ctd.):
•At the same time, they secrete periostin, a growth factor that stimulates the formation of healthy cardiac tissue.
•When the scar tissue has been digested, the bacteria bind to healthy tissue and continue secreting periostin.
•The bacteria begin to secrete Verapamil, a synthetic drug that prevents the reformation of scar tissue.
•Periodic injections of bacteria are given to the patient until scar tissue has been fully replaced by healthy tissue.
Device Diagram
Scar RepressorDevice
Scar DigestionDevice
Scar Digesting Molecule Sensor
Scar binding device
Scar digesting molecule
Trigger 2Healthy Cell
Binding Device
Trigger 1
RegeneratorDevice
Scar tissue moleculeScar digesting molecule
Scar Repressor molecule
Heart regeneration device
Bacterial Cell
B
A
C
D
E
F
G
H
I
J
K
L
M N
Timing Diagram
System Parts
Part Name Gene Name/Code (If Applicable)
HEALTHY TISSUE BINDINGDEVICE
Heterotrimeric G protein
Alpha-syntropin
Gs-alpha
Gb-y
pBAD promoter
SCAR BINDING DEVICE
SMAD 3 receptor protein
DAXX protein
HIPk2 protein
ASK-1
Map2k3
pBAD
SCAR DIGESTING SENSOR DEVICE
ASP
RBS B0034
tar/env C0082
TT B0015
NAND GATE
RBS B0034
nk022CI C0050
TT B0015
Promoter R0082
Promoter R0030
Promoter I13453
Promoter R0031
HK022CI C0050
Lambda CI C0051
OR GATE
Promoter I13453
I14032
RBS B0034
LacI C0012
TT B0015
tetR C0040
SCAR DIGESTING DEVICE
RBS B0034
Collagenase
Aspa C0083
SCAR PREVENTION DEVICE
RBS B0034
Verapamil
TISSUE REGENERATION DEVICE
RBS B0034
periostin
CHECKPOINT
EYEP E0030
morange E2050
mcherry E2060
RFP E1010
ECFP E0022
GFP I52028
ScarTissue
HealthyHeart Tissue
TGF-beta- 1
laminin Heterotrimeric G-protein
Alpha-syntropinGs-alpha
G-Beta-y
Ca 2+
Stimulatesrelease of Ca 2+ ions
Monoclonalantibody
Allosteric inhibitor
SMAD 3 DAXX
P
Phosphorylation
HIPK2 ASK-1 MAP2k3
Checkpoint 1: Enzyme-Linked Immunosorbent Assay used to detect binding of monoclonal antibody to scar tissue
Checkpoint 2: Ligand Blot Overlay and Immunoprecipitation Assays are Used to monitor the binding of proteins to one another in the scar binding and healthy tissue binding cascades
RBS
B0034
RBS
B0034
TT
B0015
Asp
Scar Digesting Sensor Device
Tar/envz
C0082
Tar/envz
C0082
Scar Binding Device
Healthy Tissue Binding Device
Parts Diagram (Left 1/3 of cell)
AspOmpRR0082
RBS
B0034
RBS
B0034
hk022CI
C0050
hk022CI
C0050
TT
B0015
pRR0050
HKCI
RBS
B0034
RBS
B0034
λ CI
C00051
λ CI
C00051
λCl
pBadI13453
RBS
B0034
RBS
B0034
hk022CI
C0051
hk022CI
C0051
TT
B0015
cI pRR0051
RBS
B0034
RBS
B0034
hk022CI
C0050
hk022CI
C0050
HKCIλCl
TRIGGER 1
I
pBad13453
RBS
B0034
RBS
B0034
lacI
C0012
lacI
C0012
TT
B0015pTet
R0040
LacI
RBS
B0034
RBS
B0034
lacI
C0012
lacI
C0012
LacI
pBad I13453
RBS
B0034
RBS
B0034
lacI
C0012
lacI
C0012
TT
B0015pLac
I14032
RBS
B0034
RBS
B0034
tetR
C040
tetR
C040
tetRlacI
TRIGGER 2
MAP2k3
Ca 2+
Ca 2+
Ca 2+
Parts Diagram (Middle 1/3 of cell)
λ CI
C00051
λ CI
C00051
λCl
hk022CI
C0050
hk022CI
C0050
HKCI
NAND Gate
lacI
C0012
lacI
C0012
LacI
tetR
C040
tetR
C040
tetR
OR Gate
mOrange
E0030
mOrange
E0030
RBS
B0034
RBS
B0034
Checkpoint 2
Glow orange
RFP
E1010
RFP
E1010
RBS
B0034
RBS
B0034
Checkpoint 4
Glow red
collagenasecollagenaseRBS
B0034
RBS
B0034
Scar digesting device
Collagenase
AspA
C0083
AspA
C0083
RBS
B0034
RBS
B0034
Asp producer
AspA
Asp
ECFP
E0022
ECFP
E0022
RBS
B0034
RBS
B0034
Reporter 1
TT
B0015
Glow cyan
TT
B0015
TT
B0015
Verapamil synthesizer
Verapamil synthesizerRBS
B0034
RBS
B0034
Scar prevention device
Verapamil
periostinperiostinRBS
B0034
RBS
B0034
Heart regeneration device
periostin
GFP
J52028
GFP
J52028
RBS
B0034
RBS
B0034
Reporter 2
TT
B0015
Glow green
Parts Diagram (Right 1/3 of cell)
Unknowns
•How will we synthesize Verapamil, a synthethic molecule? •Is there a way to incorporate a specific sequence so that the bacteriumsynthesizes Verapamil, or will this prove to be inefficient?
• How will the MAP kinase bind to pBAD?
•Is TGF-Beta specific enough of an antigen to make sure that the monoclonal antibody binds only to scar tissue and not to healthy tissue as well?
•SMAD Protein presents a similar problem. Is it specific enough?
•The protein kinases look good on paper; but will they function as predicted in vivo?
In Vivo Debugging: Fluorescent Markers
Glow orange NAND Gate “Off”
Glow cyan Scar Digesting Device “On”
Glow red
OR Gate “Off”
Glow green Scar Prevention AND Heart Regeneration Devices “On”
Reporter Debugging Function
Testing and Debugging:
Mechanism 1:• Enzyme-Linked Immunosorbent Assay used to detect binding of monoclonal antibody to scar tissue
Mechanism 2:• Immunoprecipitation Assays• Method that uses the antigen-antibody reaction principle to identify a protein that reacts specifically with an antibody from mixture of proteins so that its quantity or physical characteristics can be examined.
COSTS:
$06 typesFreePromoters
E. Coli
$10,40013 parts, 1000 bp/part
$.80 /bpDNA
$371 mg$37 /mgAntibody
Total Cost
Amount Needed
CostPart Type
Total Estimated Cost:
• Approximately $11,000• May be lower if we are able to attain DNA for the reduced iGEM price• Overall, the project appears cost efficient.
FINAL VERDICT: IT’S A GO!
References for Targeting and Bindinghttp://en.wikipedia.org/wiki/Monoclonal_antibodies
Information about monoclonal antibodies
http://www.ncbi.nlm.nih.gov/pubmed/16883602scar and healthy tissue binding
http://herkules.oulu.fi/isbn9514267214/html/x1329.htmlcardiac extracellular matrix
http://www.ncbi.nlm.nih.gov/pubmed/9521338 expression of cardiac proteins
http://www.ncbi.nlm.nih.gov/pubmed/10198196SMAD protein research
http://www.ingentaconnect.com/content/ap/mc/1999/00000031/00000003/art00902;jsessionid=cw3x1rheoz22.alice?format=print
SMAD and TGF -beta protein research
http://books.nap.edu/openbook.php?record_id=9450&page=8monoclonal antibodies
References for Targeting and Binding
http://content.nejm.org/cgi/content/full/344/23/1785engineered cardiomycotes, myocardial infarction
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T13-4CNCNXW-5&_user=501045&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000022659&_version=1&_urlVersion=0&_userid=501045&md5=b622660c2a4b7ab53b
1f770f463c8979myocardial infarction description
http://www.medicalnewstoday.com/articles/92139.phpFibroblast/tissue buildup description
References for Targeting and Binding
http://www.wipo.int/pctdb/en/wo.jsp?ELEMENT_SET=F&LANGUAGE=ENG&KEY=05%2F019471&IA=05%2F019
471&DISPLAY=DESC periostin a natural response in mice to myocardial infarctions
http://www.nature.com/nrd/journal/v6/n9/full/nrd2406.html periostin experiment showing it improves heart function after
infarctions by regenerating tissue
http://www.nature.com/nm/journal/v13/n8/abs/nm1619.html article on prospects of periostin as treatment
http://www.childrenshospital.org/newsroom/Site1339/mainpageS1339P1sublevel319.html
article on how periostin was used to induce mature heart cells to grow
