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
S O U R C E S 1.Albiniak AM, Matos CF, Branston SD, Freedman RB, Keshavarz-Moore E, Robinson C. High-level secretion of a recombinant protein to the culture medium with a Bacillus subtilis twin-arginine translocation system in Escherichia coli. The FEBS journal 2013;280:3810-21. 2.ranston SD, Matos CF, Freedman RB, Robinson C, Keshavarz-Moore E. Investigation of the impact of Tat export pathway enhancement on E. coli culture, protein production and early stage recovery. Biotechnology and bioengineering 2012;109:983-91. 3.Choi JH, Lee SY. Secretory and extracellular production of recombinant proteins using Escherichia coli. Applied microbiology and biotechnology 2004;64:625-35. 4.Gentschev I, Dietrich G, Mollenkopf HJ, et al. The Escherichia coli hemolysin secretion apparatus--a versatile antigen delivery system in attenuated Salmonella. Behring Institute Mitteilungen 1997:103-13. 5.Gusta LV, Wisniewski M, Nesbitt NT, Gusta ML. The effect of water, sugars, and proteins on the pattern of ice nucleation and propagation in acclimated and nonacclimated canola leaves. Plant physiology 2004;135:1642-53. 6.Hacker J, Neuner G. Ice propagation in plants visualized at the tissue level by infrared differential thermal analysis (IDTA). Tree physiology 2007;27:1661-70. 7.Hakim A, Nguyen JB, Basu K, et al. Crystal structure of an insect antifreeze protein and its implications for ice binding. The Journal of biological chemistry 2013;288:12295-304. 8.Hakim A, Thakral D, Zhu DF, Nguyen JB. Expression, purification, crystallization and preliminary crystallographic studies of Rhagium inquisitor antifreeze protein. Acta crystallographica Section F, Structural biology and crystallization communications 2012;68:547-50. 9.Hamed F, Fuller MP, Telli G. The pattern of freezing of grapevine shoots during early bud growth. Cryo letters 2000;21:255-60. 10.Jong WS, Sauri A, Luirink J. Extracellular production of recombinant proteins using bacterial autotransporters. Current opinion in biotechnology 2010;21:646-52. 11.Kotzsch A, Vernet E, Hammarstrom M, et al. A secretory system for bacterial production of high-profile protein targets. Protein science : a publication of the Protein Society 2011;20:597-609. 12.Kotzsch A, Vernet E, Hammarström M, et al. A secretory system for bacterial production of high-profile protein targets. Protein Science 2011;20:597-609. 13.Kristiansen E, Ramlov H, Hagen L, Pedersen SA, Andersen RA, Zachariassen KE. Isolation and characterization of hemolymph antifreeze proteins from larvae of the longhorn beetle Rhagium inquisitor (L.). Comparative biochemistry and physiology Part B, Biochemistry & molecular biology 2005;142:90-7. 14.Linton E, Walsh MK, Sims RC, Miller CD. Translocation of green fluorescent protein by comparative analysis with multiple signal peptides. Biotechnology Journal 2012;7:667-76. 15.Low K, Muhammad Mahadi N, Md. Illias R. Optimisation of signal peptide for recombinant protein secretion in bacterial hosts. Applied microbiology and biotechnology 2013;97:3811-26. 16.Lv J, Song Y, Jiang L, Wang J. Bio-inspired strategies for anti-icing. ACS nano 2014;8:3152-69. 17.Matos CF, Branston SD, Albiniak A, et al. High-yield export of a native heterologous protein to the periplasm by the tat translocation pathway in Escherichia coli. Biotechnology and bioengineering 2012;109:2533-42. 18.Mergulhão FJ, Summers DK, Monteiro GA. Recombinant protein secretion in Escherichia coli. Biotechnology advances 2005;23:177-202. 19.Moeller L, Gan Q, Wang K. A bacterial signal peptide is functional in plants and directs proteins to the secretory pathway. Journal of experimental botany 2009;60:3337-52. 20.Ni Y, Chen R. Extracellular recombinant protein production from Escherichia coli. Biotechnology letters 2009;31:1661-70. 21.Pearce RS, Fuller MP. Freezing of barley studied by infrared video thermography. Plant physiology 2001;125:227-40. 22.Perez-Perez J, Marquez G, Barbero JL, Gutierrez J. Increasing the efficiency of protein export in Escherichia coli. Bio/technology (Nature Publishing Company) 1994;12:178-80. 23.Su L, Chen S, Yi L, Woodard R, Chen J, Wu J. Extracellular overexpression of recombinant Thermobifida fusca cutinase by alpha-hemolysin secretion system in E. coli BL21(DE3). Microbial Cell Factories 2012;11:8. 24.Sugamata Y, Shiba T. Improved secretory production of recombinant proteins by random mutagenesis of hlyB, an alpha-hemolysin transporter from Escherichia coli. Applied and environmental microbiology 2005;71:656-62. 25.Taschler D, Beikircher B, Neuner G. Frost resistance and ice nucleation in leaves of five woody timberline species measured in situ during shoot expansion. Tree physiology 2004;24:331-7. 26.Thomas JD, Daniel RA, Errington J, Robinson C. Export of active green fluorescent protein to the periplasm by the twin-arginine translocase (Tat) pathway in Escherichia coli. Molecular microbiology 2001;39:47-53
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
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