session 4 #3 ajo-franklin engineering thermophilic ...€¦ · engineering thermophilic...
TRANSCRIPT
Engineering ThermophilicMicroorganismsMicroorganisms for Mineral Recovery
Caroline Ajo Franklin Caroline Ajo-Franklin Staff Scientist, Berkeley Lab cafgroup.lbl.gov 200 nm
California Geothermal Forum October 20, 2016
My laboratory’s research
Interfacing living cells & materials
CAF, Noy. Advanced Materials (2015).TerAvest, CAF. Biotechnology & Bioengineering (2016).Zhuang, CAF, et al. Current Opinion in Biotechnology (2015).
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introduction
Need robust, selective metal binding under hot, salty conditionsunder hot, salty conditions
Metal ~Conc. (mg/kg H O)H2O)
Na+ 71,000Ca2+ 11,000K+ 4,500
Mg2+ 2,000Zn2+ 100
Gadd Pazirandeh et al. Applied & Environmental Biotechnology (1995).
Gd3+ 10x10-3
Can we combine robustness with selectivity?pp & gy ( ) Park, Jiao et al. Environmental Science & Technology (2016).
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Can we combine robustness with selectivity?
Introduction
Thermophilic S-layers provide a robust display approachp y pp
50nm
10µm
Moll et al. Proceeding of the National Academies of Sciences, USA (2002)
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St t S l ff ld d t l bi diresults
Strategy: S-layer scaffolded metal binding domains
Protoplasted cells
10µm
Thermophilic host:Geobacillus stearothermophilus
Robust scaffold:SbsB s-layer
Selective binding:Metal-binding domain
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results
We identified criteria to get 90% pure Zn2+, Gd3+
Metal ~Conc. (mg/kg H O)
Zn2+Gd3+
90% pure
H2O)
Na+ 71,000Ca2+ 11,000K+ 4,500
Mg2+ 2,000Zn2+ 100
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Kd for Gd3+ < 10 nMKd for Zn2+ < 30 µM
Gd3+ 10x10-3 Kd Zn2+
Kd Co2+>10
Kd Gd3+
Kd Ca2+ >105
Charrier, CAF, in prep.
results
We designed thermophilic S-layers to bind specific metalsp
Monomeric E. coli Production PlasmidFusion Proteinhost
+
Plasmid
+ =
1 Zn domain1. Zn domain.2. Double gadolinium domain.
Charrier, CAF, in prep.
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results
Engineered S-layers self-assemble into nanosheets
Native s-layer nanosheet
+Ca2+
Engineered s-layer nanosheet
+Ca2+
Charrier, CAF, in prep.
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results
Engineered S-layers self-assemble into nanosheets
Native s-layer nanosheet
+Ca2+
Engineered s-layer nanosheet
+Ca2+
200nm
Charrier, CAF, in prep.
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results
Engineered nanosheets bind Zn2+
with higher affinity than nativewith higher affinity than native
Isolated binding domain:1 Zn2+ per binding domainKd = 0.14 nM
Charrier, CAF, in prep.
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results
At 40ºC, engineered nanosheets bind Zn2+
with higher affinitywith higher affinity
E ’d
Displayed binding domain:~ 4 Zn2+ per binding domain
Eng’dnanosheet
p g
Nativenanosheet
Charrier, CAF, in prep.
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results
Engineered nanosheets bind Zn2+
more selectivelymore selectively
Analog brine solution: KCl, CaCl, & NaCl
Engineered nanosheet
Displayed binding domain:~1 6 Zn2+ per binding domain
Nativenanosheet
1.6 Zn per binding domain
Charrier, CAF, in prep.
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results
At 40ºC, engineered nanosheets bind Gd3+
more selectivelymore selectively
Engineered nanosheet
Displayed binding domain:~0.7 Gd3+ per binding domain
Nativenanosheet
Charrier, CAF, in prep.
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summaryy
Surface-layer proteins can be used to create robust materials that bind with biological-specificity.
Engineered nanosheets have higher affinity, selectivity for their metal of interest than native nanosheets.
Ca2+
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I i i f th lOn-going work
In vivo expression for geothermal applications
Protoplasted cells
10µm
Thermophilic host:Geobacillus stearothermophilus
Robust scaffold:SbsB s-layer
Selective binding:Metal-binding domain
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Future work
Differing melting temperatures could provide a unique level of control.p q
40°C 70°C
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summary CAF lab
acknowledgements Dr. Moshe Baruch Dr. Michaela TerAvest Marimikel Charrier Dr. Tatsuya Fukushima Dr. Behzad Rad Matt Hepler Dr. Heather Jensen Dr. Jenny Cappuccio Dr. Li Yang Andrew Prior, Joshua Deng Greg Murray Albert Shon, Nicole Beedle, Kostia
Funding:
Malley, Mark Kokish, Annie Li, Dimitri Umpriani
Collaborators:Dr. Jonathan Ajo-FranklinProf. Lars Angenent, CornellDr. Vivek Mutalik, LBLDr. Heinz Frei, LBLDr. Corie Ralston, LBLProf. Adam Arkin, UC-BerkeleyProf. Michel Maharbiz, UC-Berkeley
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Thank you!
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