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HUST-CHINA BACKGROUND A Novel Method Antihypertensive Drug - - PowerPoint PPT Presentation
HUST-CHINA BACKGROUND A Novel Method Antihypertensive Drug - - PowerPoint PPT Presentation
Circadian Release of Propionate for Antihypertensive Therapy HUST-CHINA BACKGROUND A Novel Method Antihypertensive Drug Engineered Bacteria Interacts with Olfr78, a GPCR expressed in smooth muscle cells of Propionate small blood
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Engineered Bacteria Antihypertensive Drug
A Novel Method
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Jennifer L. Pluznick, et al. PNAS, 2012, 11, 4410–4415
Propionate
Interacts with Olfr78, a GPCR
expressed in smooth muscle cells of small blood vessels
Induce vasodilatation and
hypotension
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A Novel Method
Engineered Machine
Propionate Oscillator
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Our Progresses
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Background
Wood-Werkman Reaction
Haller T, Buckel T, Rétey J et al. Biochemistry, 2000, 39(16): 4622-46229.
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Plasmids Construction
- Insert four genes into
pET-28a(+) plasmids
- Transform them into
BL21 E.coli strains
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Protein Expression
Marker sbm ygfD ygfG ygfH Control Successfully validate the expression of sbm, ygfD, ygfG, and ygfH. 170 130 100 70 55 40 35 25 15 (KDa)
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22787.7 23810.4 24051.3 24986.3 25765.1 20000 21000 22000 23000 24000 25000 26000 27000
4.49% 9.41% 9.65% 13.07%
**
Concentration of propionate (mg/L)
n.s. n.s. n.s.
P<0.01
Control ygfH ygfG sbm ygfD
HPLC analysis of wild-type BL21 and recombinant BL21 with four genes (4hrs after IPTG induction)
Production Increasement
Propionate's output can be increased over 10% with ygfD
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Question How can we make ygfD expressed periodically?
OSCILLATOR !
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Our Progresses
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Hybrid Promoter
Activation
from araBAD promoter
Repression
from lacZYA promoter
+Arabinose +AraC
- IPTG
+LacI Hybrid Promoter
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+Arabinose +IPTG
lacl
Hybrid Promoter
araC
Hybrid Promoter
Oscillator
- IPTG
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Issues
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The Single-Cell Level
Delay Differential Equations (DDEs) AraC’s oscillation, τ = 30min, T = 11 hours
τ ~N(30.0,1.02)min T1 = 644.8min, T2 = 639.2min, T3 = 654.1min, T4 = 642.6min, T5 = 645.1min
Random Period
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The Multi-Cell Level
A group of genetic oscillating cells can oscillate as well as a single one does.
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Adjusting Period
The period range is limited to 44min – 50min when altering Arabinose and IPTG, with τ = 2 min.
Not Satisfying
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Period increases linearly and unlimitedly when delay τ increases.
Adjusting Period
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Achieve rapid protein degradation Reporter pET-28a(+) +Arabinose +IPTG
Oscillator Construction
LAA
lacl
Hybrid Promoter LAA
araC
Hybrid Promoter LAA
mRFP pACYCDuet-1
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Immobilized cell with oscillator expresses RFP periodically
LAA
araC mRFP
Hybrid Promoter LAA
Validation of constructed pET-28a(+)
Validation of Oscillator
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Validation of Oscillator
Fluorescence density of multi- cells changes in an
- scillating way
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Wet-Lab & Modeling
Wet-lab work and modeling results consistent with each
- ther well.
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Our Progresses
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Future Plan
Circadian release of high-level propionate Replace mRFP with ygfD
LAA
araC mRFP Hybrid Promoter
LAA LAA
araC mRFP Hybrid Promoter
LAA
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- Regulating the period of propionate release under the direction of modeling
- Mathematically simulate the “in vivo” environment
Modeling
Future Plan
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Transfer the engineered machine into Bifidobacterium Bifidobacter dobacteriu ium E.col coli
Future Plan
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Human Practice
A Lecture In The High School Collaborations Crosswords Game
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Human Practice
A Talk to The Public Questionnaire
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Accomplishments
I. Subcloned sbm, ygfG, ygfH and ygfD, constructed and documented the expression vectors of the four genes II. Constructed the oscillator with mRFP and LAA tags
- III. Found the gene that can realize the
highest production of propionate
- IV. Achieved the periodical fluorescence
intensity simulated working process of the oscillator V. Analyzed the sensitivity and the robustness of the oscillator, providing ways to adjust for a wanted period
- VI. Verified that oscillators in multi cells
can oscillate as well as in a single one
- VII. A new application in hypertension
treatment VIII.Shared information with WHU & HZAU, and help HZAU complete a biobrick
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Acknowledgement
Guangdong Shang Jingnan Lu Jeff Hasty Roya Mahmoudi
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We are HUST-China
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