P L A N T I F R E E Z E CSW ProTeens 2014 T H E P R O B L E M T - PowerPoint PPT Presentation

P L A N T I F R E E Z E CSW ProTeens 2014

T H E P R O B L E M

T H E P R O B L E M • Early frosts present challenges to agrobusiness: damaging crops and lowering profits ($1 billion) • Climate change brings earlier cold weather and more random frosts

L O W - T E C H S O L U T I O N S • Application of water to fields before frost • Smudge pots • Wind machines

F R O S T B A N • A biological solution was proposed in 1980s by Steven E. Lindow. • Mutated versions of the bacteria P. syringae • Plants with P. syringae on surface were much less susceptible to frost damage. • Its use as an anti-frost device in food crops brought up enormous backlash among critics. • Never commercialized

J U S T I F I C A T I O N

J U S T I F I C A T I O N ● No effective, safe solutions ● Naturally occurring antifreeze proteins (AFPs) are over 300x more effective than conventional chemical antifreezes at same concentrations ● AFPs already used industrially (Breyers Ice Cream)

J U S T I F I C A T I O N: Why RiAFP? ● Most AFPs used are from fish ● Insect proteins 100x more active ● RiAFP (AFP from the Rhagium inquisitor beetle) is the most potent known AFP ● Harder to harvest at large scale from insects

J U S T I F I C A T I O N: Why synthesize AFP in E.coli ? ● Engineered organisms could produce hard-to harvest, more effective insect AFPs ● Synthetic bio solution would combine benefits of AFPs: efficient production of most efficient protein ● AFP long-term storage not necessary, easily inducible production

S O L U T I O N

S O L U T I O N: PlantiFreeze A genetically engineered E. coli that secretes non-toxic RiAFP applied to the exterior of plants, inhibiting ice-crystal formation.

D E S I G N A dual-plasmid system: Expression Plasmid (for expressing RiAFP) pLG575 (allows tagged RiAFP secretion)

E X P R E S S I O N P L A S M I D ● T7-RiAFP/His/HlyA/double terminator ● New plasmid!

p L G 5 7 5 S E C R E T I O N P L A S M I D ● Codes for HlyB, HlyD, and TolC. ● Allows HlyA-tagged proteins to be secreted via the HlyB- HlyD-TolC translocator.

M E T H O D S

D I R E C T S Y N T H E S I S ● Knew the DNA sequence. 864 bp. ● Inexpensive ● Reliable by IDT

P L A S M I D M A P P I N G ● SeqBuilder Software ● Checked ORFs and Restriction Sites

A S S E M B L I N G P L A S M I D ● EcoRI and PstI ● pSB1A3 linearized backbone ● Ampicillin Resistance

N A N O D R O P O F E X P R E S S I O N P L A S M I D

E L E C T R O P H O R E S I S O F E X P R E S S I O N P L A S M I D KprI + HindIII EcoRI + PstI DNA LADDER 1 2 3 4 5 6 7 8 9 10 11 12 k b 12,000 ➔ 850 ➔ 100 ➔

S E C R E T I O N P L A S M I D ● pLG575 ● Almost 100 ng/uL ● Chloramphenicol Resistance

C O - T R A N S F O R M A T I O N ● Compatible plasmids: different origins of replication ● Different antibiotic resistance ● Plate with amp + CM to select for both ● After reaching higher concentrations of plasmid, we had growth!

G R O W T H O N A M P + C M

I N D U C T I O N ● IPTG Induction of RiAFP production ● Constitutive promoter for pLG575 secretion plasmid ● 12 hour wait time

D E T E C T I O N O F R i A F P

E X P E R I M E N T A L T I M E L I N E Time 0 hours 12 hours 24 hours IPTG induction Cells put in of half LB broth

E L E C T R O P H O R E S I S Wells ran in the gel: • Pre-induction supernatant • Pre-induction lysate • Post-induction supernatant • Post-induction lysate • His-tagged molecular weight benchmark

S T A I N I N G ● InVisionTM His-tag In-gel Stain ● Used a UV transilluminator equipped with a camera

R E S U L T S

F U T U R E I D E A S

F U T U R E I D E A S • Optimizing transformation efficiency • Inducing biofilm formation • Incorporating a kill switch • Temperature sensitive promoter • Consider non-agricultural applications

S P O N S O R S

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T H A N K Y O U!

H O W A N T I - F R E E Z E P R O T E I N S W O R K I c e C r AFP y s t a l AFPs bind to non-basal planes of ice, inhibiting thermodynamically favored ice growth.

C R Y S T A L L I Z A T I O N I N H I B I T I O N

H U M A N P R A C T I C E S ● Illuminarium ● presenting to school ● teaching about iGEM & our project