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 vessels Induce vasodilatation and hypotension Jennifer L. Pluznick, et al. PNAS, 2012, 11, 4410 – 4415
A Novel Method Propionate Oscillator Engineered Machine
Our Progresses
Background Wood-Werkman Reaction Haller T, Buckel T, Rétey J et al. Biochemistry, 2000, 39(16): 4622-46229.
Plasmids Construction • Insert four genes into pET-28a(+) plasmids • Transform them into BL21 E.coli strains
Protein Expression Successfully validate the expression of sbm, ygfD, ygfG, and ygfH. (KDa) Marker sbm ygfD ygfG ygfH Control 170 130 100 70 55 40 35 25 15
Production Increasement Propionate's output can be increased over 10% with ygfD HPLC analysis of wild-type BL21 and recombinant BL21 with four genes (4hrs after IPTG induction) Concentration of propionate (mg/L) 27000 ** P<0.01 n.s. 26000 25765.1 n.s. 25000 n.s. 24986.3 24000 24051.3 23810.4 23000 22787.7 9.65% 13.07 % 4.49% 9.41% 22000 21000 20000 Control ygfH ygfG sbm ygfD
Question How can we make ygfD expressed periodically? OSCILLATOR !
Our Progresses
Hybrid Promoter +Arabinose -IPTG Hybrid Promoter +AraC +LacI Activation Repression from araBAD from lacZYA promoter promoter
Oscillator +Arabinose +IPTG Hybrid Promoter araC - IPTG Hybrid Promoter lacl
Issues
The Single-Cell Level Delay Differential Equations (DDEs) AraC’s oscillation, τ = 30min, T = 11 hours Random Period τ ~N(30.0,1.0 2 )min T 1 = 644.8min, T 2 = 639.2min, T 3 = 654.1min, T 4 = 642.6min, T 5 = 645.1min
The Multi-Cell Level A group of genetic oscillating cells can oscillate as well as a single one does.
Adjusting Period Not Satisfying The period range is limited to 44min – 50min when altering Arabinose and IPTG, with τ = 2 min.
Adjusting Period Period increases linearly and unlimitedly when delay τ increases.
Oscillator Construction Achieve rapid protein +Arabinose +IPTG degradation pACYCDuet-1 Hybrid Promoter araC mRFP LAA LAA Reporter Hybrid Promoter lacl LAA pET-28a(+)
Validation of Oscillator Validation of constructed pET-28a(+) araC mRFP Hybrid Promoter LAA LAA Immobilized cell with oscillator expresses RFP periodically
Validation of Oscillator Fluorescence density of multi- cells changes in an oscillating way
Wet-Lab & Modeling Wet-lab work and modeling results consistent with each other well.
Our Progresses
Future Plan Hybrid Promoter araC mRFP LAA LAA Replace mRFP with ygfD Hybrid Promoter araC mRFP LAA LAA Circadian release of high-level propionate
Future Plan • Regulating the period of propionate release under the direction of modeling • Mathematically simulate the “ in vivo ” environment Modeling
Future Plan Transfer the engineered machine into Bifidobacterium E.col coli Bifidobacter dobacteriu ium
Human Practice A Lecture In The High School Crosswords Game Collaborations
Human Practice Questionnaire A Talk to The Public
Accomplishments I. Subcloned sbm, ygfG, ygfH and ygfD, V. Analyzed the sensitivity and the constructed and documented the robustness of the oscillator, providing expression vectors of the four genes ways to adjust for a wanted period II. Constructed the oscillator with mRFP VI. Verified that oscillators in multi cells and LAA tags can oscillate as well as in a single one III. Found the gene that can realize the VII. A new application in hypertension highest production of propionate treatment IV. Achieved the periodical fluorescence VIII.Shared information with WHU & HZAU, intensity simulated working process of and help HZAU complete a biobrick the oscillator
Acknowledgement Guangdong Shang Jingnan Lu Jeff Hasty Roya Mahmoudi
We are HUST-China
Circadian Release of Propionate for Antihypertensive Therapy HUST-CHINA
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