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Presented by
Leo Seak Chi U(Team leader)
Ricky Chan Wai Lun(Wet lab team)
Joe Ho Cho Tai(Modeling team)
Elaine Chiu Yee Ting(Human Practice team)
1
AzotoBacter vinelandii Cluster-transformable
& Deoxygenated protein Expression system
(ABCDE system)
2
ABCDE System
Introduction
3
Problem: Energy and Environment
𝐶𝑂2 𝐶𝐻4nitrogenase
Hydrogenase III (H2ase) ADP Nitrogenase(N2ase)
H2
H+
Ferredoxin reduced
Ferredoxin oxidized
ATP
Fe protein oxidized
Fe protein reduced
MoFe protein reduced
MoFe protein oxidized
CH4
CO2
Supported by Guiral et.al.,2005 5
Mechanism
Byproduct of nitrogenase
Needed for CH4
production𝐶𝑂2
Problem of Introducing Nitrogenase
•Oxygen sensitive (degenerated by oxygen)
•Anaerobic condition is needed
6Zhi-Yong Yang et al., PNAS 2012
2013 Bielefeld-Germany iGEM team 2013 Copenhagen iGEM team
7
Previous iGEM Team
AzotoBacter vinelandii Cluster-transformable &
Deoxygenated protein Expression system
(ABCDE system)
8
Solution: ABCDE System
Why Azotobacter vinelandii
Azotobacter vinelandii (strain DJ)
June 2014
• Intracellular anaerobic environment
• Stable genome integration
• Safety level: group 1 bacteria
• nifH promoter
(ammonium-repressible)
Sabra, W., Zeng, A. P., Lünsdorf, H., & Deckwer, W. D. (2000)Applied and environmental microbiology, 66(9), 4037-4044.
AzotoBacter vinelandii Cluster-transformable &
Deoxygenated protein Expression system (ABCDE system)
10
Biobricks and CharacterizationAmmonium-Repressible T7 Protein Expression System
11
ABCDE: Basic Parts
Parts Description
BBa_K1314000 Ammonia repressible nifH promoter
BBa_K1314002 150bp random sequence (sequence for homology)
BBa_K1314003 part of mRFP(sequence for homology)
BBa_K1314012 nif K homologous sequence
Status
Submitted and accepted
Submitted and accepted
Not submitted
Not submitted
12
Characterization of BBa_K1314000: nifH Promoter
transcriptional regulation mechanism of
nifHDK operon in A. vinelandii
Hamilton, T. L., Ludwig, M., Dixon, R., Boyd, E. S., Dos Santos, P. C., Setubal, J. C., ... &
Peters, J. W. (2011). Transcriptional profiling of nitrogen fixation in Azotobacter vinelandii.
Journal of bacteriology, 193(17), 4477-4486 13
Characterization of BBa_K1314000: nifH Promoter
300bp
200bp
100bp
Gel photo of EcoRI PstI double digest
check of BBa_K1314000
nifH regulated RFP expression(left) versus
constitutive RFP expression(right) in DH5α
14
nifH promoter
Parts Description
BBa_K1314001 nifH promoter with mRFP reporter
BBa_K1314004 Ptet mRFP with random sequence
BBa_K1314005 Ammonia- repressible T7 RNA polymerase generator(strong)
BBa_K1314006 Ammonia- repressible T7 RNA polymerase generator(weak)
BBa_K1314007 Ammonia- repressible T7 RNA polymerase generator(strong) with ptet mRFP reporter
BBa_K1314008 Ammonia- repressible T7 RNA polymerase generator(weak) with ptet mRFP reporter
BBa_K1314009 Amilcp reporter with part of rfp
BBa_K1314010 T7 promoter with random sequence
ABCDE: Composite Parts
Status
Submitted and accepted
Submitted and accepted
Submitted and accepted
Submitted and accepted
Submitted and accepted
Submitted and accepted
Submitted and accepted
Submitted and accepted15
ABCDE: Composite Parts
Composite PartsParts Description Status
BBa_K1314010 T7 promoter with random sequence Submitted and accepted
BBa_K1314011 T7 promoter amilcp reporter with homologous sequence
Submitted and accepted
BBa_K1314013 Ammonia- repressible T7 RNA polymerase generator(strong) with ptet mRFP reporter( site-specific integration version)
Submitted and accepted
BBa_K1314014 Ammonia- repressible T7 RNA polymerase generator(weak) with ptet mRFP reporter( site-specific integration version)
Submitted and accepted
16
BBa_K1314013/14: T7 RNA Polymerase into A.vinelandii
Reciprocal recombination in A.vinelandii
Patricia Dos Santos, Molecular Biology and Genetic Engineering in Nitrogen Fixation17
Foreign DNA as a
Vector
Wild type A.vinelandii
genome
Mutated A.vinelandii
genome
Circular DNA of BBa_K1314013 with Ampicillin resistance gene 18
BBa_K1314013/14: Making the Vector
Linearized DNA of BBa_K1314013 with Ampicillin
resistance gene by cutting XhoISingle
Digestion
using XhoI
BBa_K1314013/14: Modifying the A. vinelandii genome
genomic sequence of nifHDK operon in wild type A. vinelandii
19
Linearized DNA of BBa_K1314013 with
Ampicillin resistance gene by cutting XhoI
Hamilton, T. L., Ludwig, M., Dixon, R., Boyd, E. S., Dos
Santos, P. C., Setubal, J. C., ... & Peters, J. W. (2011).
Transcriptional profiling of nitrogen fixation in Azotobacter
vinelandii. Journal of bacteriology, 193(17), 4477-4486.
Characterization of BBa_K1314013/14
5000bp
Gel electrophoresis of EcoRI
PstI double digestion check of
BBa_K1314013/14
20
Characterization of BBa_K1314013/14
Ammonium replete condition
BBa_K1314014(no growth) BBa_K1314013
(no growth)
BBa_K1314001(RFP
expression but poor
growth)
Wildtype
A.vinelandii
Normal nitrogenase
expression
High RFP expression
High RFP expression
well grown but no red RFP
expression
21Ammonium depleted condition
Introduce gene under T7 promoter into A.vinelandii
Strain Colour change from red to blue
Part of genome sequence of A.vinelandii with BBa_K1314013 integrated
Sequence of DNA Vector BBa_K1314011 22
Characterization of BBa_K1314011
BL21 BFP expression of K1314011(right,10mM IPTG added) and negative control(left, no IPTG added) 23
BBa_K1314015: Vector for insertion of desired protein for T7 expression
Plasmid Backbone
3000bp
2000bp
EcoRI PstI double digest check gel photo
Status
Submitted (Kanamycin resistance)
24
~2500bp insert
Description
Part:BBa_K1314015 Homology integration toolkit
Genome Integration Has Never Been Easier
Plasmid map of
BBa_K1314015
Quick method for making desired vector
25
Restriction + Ligation = Vector for integration
Modeling
26
ABCDE (Protein Expression)-solve the contradiction
ABCDE system (Methane Energy)- rate & efficiency
Problem in protein expression
27
ɑ-RNA
polymerase
nifH
promoter T7polymeraseT7 promoter Target
proteins
repressed by ammonia
needs ammonia
ABCDE (Protein Expression)-solve the contradiction
Solution: Separate into two stages
Stage one
Producing T7
polymerase
28
Stage two
Producing target
proteins
𝑁𝐻3𝑁𝐻3
𝑁𝐻3
𝑁𝐻3
𝑁𝐻3
𝑁𝐻3
ABCDE (Protein Expression)-solve the contradiction
We have the following equation for the rate of change of [N]:
𝑑[𝑁]
𝑑𝑡=-A
𝑑[𝑇7]
𝑑𝑡-B
𝑑[𝑇𝑃]
𝑑𝑡−
𝐶[𝑁]
𝐷+[𝑁]
• A: nitrogen needed to expression one T7 polymerase;
• B: nitrogen needed to expression one target protein;
• C: maximum speed of nitrogen consumption of unmodified bacteria;
• D: concentration needed for the rate of nitrogen consumption equals to half of maximum speed.
29
ABCDE (Protein Expression)-solve the contradiction
Stage one: producing T7 polymerase
Reference:Michaelis, L.; Menten, M.L. (1913). "Die Kinetik der Invertinwirkung". Biochem Z 49: 333–369
30
𝑁𝐻3
𝑑[𝑇7]
𝑑𝑡= 𝐸[𝑁]𝑒−𝐹[𝑁]
ABCDE (Protein Expression)-solve the contradiction
Stage one: producing T7 polymerase
Stage two: producing target protein
31
ABCDE (Protein Expression)-solve the contradiction
𝑁𝐻3𝑁𝐻3𝑁𝐻3𝑁𝐻3
𝑁𝐻3
𝑑[𝑇𝑃]
𝑑𝑡= 𝑉𝑚𝑎𝑥( 𝑇7 )
0
1
2
3
4
5
6
7
8
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2
Co
nce
ntr
atio
n
time
Analysis: rate equations for CH4 production
Reference: 1.G. P. Moss(1981)” Symbolism and Terminology in Enzyme Kinetics"http://www.chem.qmul.ac.uk/iubmb/kinetics/2. Michael W.W. Adams(1990)” The structure and mechanism of iron-hydrogenases “Biophysica Acta, 1020 (1990) 115-145
𝑑[𝐶𝐻4]
𝑑𝑡= 𝐾1 𝐻+ [𝐶𝑂2]=𝐾1𝐾2 𝐻+ = 𝐾 𝐻+
𝑑[𝐻+]
𝑑𝑡= 𝐾3[𝐻2][oxidized ferredoxin]-k’’
𝑑[𝐶𝐻4]
𝑑𝑡
=𝐾3𝐾4 𝐶𝐻4 [oxidized ferredoxin] -k’’𝑑[𝐶𝐻4]
𝑑𝑡
=𝐾3𝐾4𝐾5 𝐶𝐻4 −k’’𝑑[𝐶𝐻4]
𝑑𝑡
=K’ 𝐶𝐻4 -k’’𝑑[𝐶𝐻4]
𝑑𝑡
𝐾1 = rate constant; 𝐾2 = 𝐶𝑂2𝑛; K = K1K2;
𝐾3= rate constant; [𝐻+]=𝐾4 𝐶𝐻4 ; [oxidized ferredoxin]=𝐾5; K’ = K3K4K5. K’’=𝐾4+8
ABCDE system (Methane Energy)- rate & efficiency
33
ABCDE system (Methane Energy)- rate & efficiency
[𝐶𝐻4] = 𝑐1exp 𝑐2𝑡 + 𝑐3exp(𝑐4𝑡)
Result: Energy Efficiency
34
ABCDE system (Methane Energy)- rate & efficiency
Energy Output (𝐸𝑜𝑢𝑡𝑝𝑢𝑡):
𝐸𝑐: combustion energy of CH4
Energy input (𝐸𝑖𝑛𝑝𝑢𝑡):
𝐸1: ATP energy for enzyme activity – producing 𝐶𝐻4
𝐸2: ATP energy for producing 𝐻2
𝐸𝑚: Metabolic energy
𝐸𝐸: Environmental consumption
Energy Efficiency = = = 5.36 % 𝐸𝑜𝑢𝑡𝑝𝑢𝑡
𝐸𝑖𝑛𝑝𝑢𝑡
𝐸𝑐
𝐸1+𝐸2 + 𝐸𝑚+𝐸𝐸
16 ATP2 ATP
𝐶𝑂2
𝐶𝐻4+2𝐻2𝑂𝐻2
~ 2 times of photosynthesis in crop plants(2%)Reference: Govindjee, What is photosynthesis?
Human Practice and Safety
35
Collaboration
Cooperate with CityU_HK, Hong_Kong_HKUST, Berlin
-Cloning
-Modelling
-Ideas exchanging
Material Exchanging Platform
36
Collaboration
37
BBa_K1472614(3384bp)
Invitrogen 1kb plus ladder
3000bp4000bp
~3600bp
Colony PCR with biobricksequencing primer
What do people think about our project
38
Do you think this project is beneficial to the human being?
39
0
10
20
30
40
50
60
70
80
90
100
Yes No No opinion
HS (102) UG (408) PG (61) Other* (32)
%
HS: High SchoolUG: Under-graduatePG: Post-graduateOther: Any responders other than these three
What is the most attractive highlight to you in this project?
40
%
0102030405060708090
Environmentalfriendly
Bacterial-based Creativity Practical None Other
HS (102) UG (408) PG (61) Other* (32)HS: High SchoolUG: Under-graduatePG: Post-graduateOther: Any responders other than these three
(Interesting)
In which aspect do you think this project is helpful and therefore worth-conducting?
41
0
10
20
30
40
50
60
70
Energy Crisis Global Warming None Other
HS (102) UG (408) PG (61) Other* (32)
%
HS: High SchoolUG: Under-graduatePG: Post-graduateOther: Any responders other than these three
What will be your main concern of this project?
42
0
10
20
30
40
50
60
70
80
90
Safety Efficiency Cost Other Not at all
HS (102) UG (408) PG (61) Other* (32)
%
HS: High SchoolUG: Under-graduatePG: Post-graduateOther: Any responders other than these three
Safety
• Lab safety training course
• Proper handling of biological and chemical materials
• Proper disposal of biological and chemical waste
• Personal protective equipment
• Only organisms in risk group 1 are used
• Negative pressure laboratory
• Communication with supervisors regularly
43
Workshops
Laboratory Demonstration
-Introduce simple lab tools
-Some simple cloning techniques
-*Focus on increasing safety awareness
44
Have you heard of International GeneticalEngineered Machine(iGEM) or Synthetic Biology?
45
0
20
40
60
80
100
Neither iGEM only Synthetic Biology Only Both
HS (102) UG (408) PG (61) Other* (32)
%
HS: High SchoolUG: Under-graduatePG: Post-graduateOther: Any responders other than these three
Do you think there are enough ways for public to reach Synthetic Biology?
46
0
10
20
30
40
50
60
70
80
90
100
Yes No Not sure
HS (102) UG (408) PG (61) Other* (32)
%
HS: High SchoolUG: Under-graduatePG: Post-graduateOther: Any responders other than these three
In your point of view, which is the most effective way to raise public awareness towards synthetic biology?
47
0
10
20
30
40
50
60
70
Leaflet Poster Workshop Other
HS (102) UG (408) PG (61) Other* (32)
%
HS: High SchoolUG: Under-graduatePG: Post-graduateOther: Any responders other than these three
(Banner)
Outreach
University
Art Fair
Energy Day Collaboration
High School
WorkshopsCUHK
Orientation Day
Industry
Energy Day
International
Poster Collaboration
48
Outreach
University
Art Fair
Energy Day Collaboration
High School
WorkshopsCUHK
Orientation Day
Industry
Energy Day
International
Poster Collaboration
49
Energy day
Poster presentation
-Promote our project
-Receive professional comments
There are industries interested in our project
50
Outreach
University
Art Fair
Energy Day Collaboration
High School
WorkshopsCUHK
Orientation Day
Industry
Energy Day
International
Poster Collaboration
51
Art fair, CUHK Orientation Day, Workshops & Poster Exhibition
Poster Exhibition
-Promotion of iGEM
and Synthetic Biology
-Promotion of Project
-Recruitment of next year iGEM team
Leaflet Distribution
52
Would you join activities related to Synthetic Biology (say, iGEM), if you were given a chance?
53
0
10
20
30
40
50
60
70
80
90
100
Yes No
HS (102) UG (408) PG (61) Other* (32)
%
HS: High SchoolUG: Under-graduatePG: Post-graduateOther: Any responders other than these three
More!
• First intracellular anaerobic protein expression system
• Introduce Azotobacter vinelandii to iGEM
• Try to make registry biobricks usable in the chassis
• Carbon fixation with the bacteria
54
Bronze Completed Judging form
Wiki
Sliver Characterize Bba_K1314000,
Bba_K1314001, Bba_K1314011,
Bba_K1314013, Bba_K1314014
16 biobricks submitted and accepted
Gold Improved and characterized E1010 and
K529009
Helped CityU to construct a functional
biobrick with some characterization
Analyzed difference between postgraduate,
high school and college student
Linking all the iGEM together by material
changing platform
Medal?
• Electricity production
• Magnetosome production
• Carbon fixation
• Nitrogen fixation
• ……More to be discovered
55
ABCDE is just a beginning!
And Further More…
Acknowledgement
Our Sponsors:
56
Special Acknowledgement:• Professor CHAN King Ming (Associate Professor, School of Life Sciences)
• Professor CHAN Ting Fung (Associate Professor, School of Life Sciences)
Thank you both for giving advices and guidance throughout the project.
• Professor Pun To, Douglas YUNG (Assistant Professor)
• Mr. Nelson So (Technician - BME Teaching Lab)
Thank you both for providing place and support to the project.
• Professor Jimmy C. M. YU (Professor of Chemistry)
Thank you for giving suggestions of the project.
• Thank you Professor Dennis R. Dean and Ms. Valerie Cash for giving DNA and strain dj of Azotobactor Vinlandii.
• Thank you Dr. Marianne Guira for giving frozen pellet of Aquifex Aeolicus.
• Thank you Dr. Zhu Yuan (Dept. of Biology and Chemistry, City University of Hong Kong) for modelling work.
57
Q&A
Thank You!
58