Solar pMFC - 2015.igem.org2015.igem.org/files/poster/UNITN-Trento.pdf · 2015-10-08 · Team:...
1
Team: Alessadro Turcato, Claudio Oss Pegorar, Elisa Godino, Moreno Zolfo, Riccardo Corbellari, Silvia Galvan, Veronica Pinamon. Special Thanks to: Solar pMFC UNITN-Trento A microbial fuel cell with a light driven E. coli engine MORE ATP WITH PROTEORHODOPSIN. 0 5 10 15 20 BBa_K731201 BBa_K1604010 Dark Dark Light Light araC-pBAD Proteorhodopsin ADP/ATP ratio araC-pBAD BBa_K731201 Proteorhodopsin BBa_K1604010 ADP/ATP ratio Proteorhodopsin (PR) [1] creates a light-driven proton gradient, activating ATP synthase and increasing bacterial lifespan [2] . NEB10β expressing BBa_K1604010 (proteorhodopsin) were placed in the anaerobic anodic chamber of a small MFC togheter with chemical mediators (anode, blue methylene 100 uM; cath- ode ferricyanide 10 mM). OUR BACTERIA ARE HAPPY IN THE MFC! WE HAVE MORE NAD + pncB [3] catalyzes the rate limiting step of NAD + biosynthesis. NAD + /NADH ratios were measured with a colorimetric assay, in an- aerobic conditions. BBa_K1604031 (pncb, yellow) and negative control BBa_K731201 (aracpBAD, green). Our Solar pMFC under costruction (left) and at work (right). It is composed of 6 cathods in parallel that share the same anodic chamber. We obtained a maximum power of 109 μW with a cur- rent of 467 μA (external resistance of 500 Ω). We succesfully produced β-carotene (B- Ba_K1604020) which is cleaved by blh in two molecules of retinal (BBa_K1604022) in NEB10b cells. WHERE IS OUR RETINAL? ATTRIBUTIONS: Instructors: Cristina del Bianco, Sheref S.Mansy, Olivier Jousson, Martin Hanczyc Proteorhodopsin: S.G, R.C, E.G, blh; V.P, pncB: R.C, Mtr: A.T, MFC: A.T, R.C, Interlab: E.G, C.O.P, wiki master M.Z. Human practices: E.G. Prototype: A.T, R.C with the support of the Physicis Dept. workshop and Damiano Avi, Mariano di Mauro and Gigi Minati for helpful suggestions. Carolyn Ajo Franklin for bacteria with Mtr system. Martin Hanczyc for providing MFC materials. Graziano Guella for access to the HPLC. Gian Marco Franceschini for helping with the logo and graphics. The Mansy lab for some help in interlab study. REFERENCE: Proteorhodopsin and blh [1] Martinez, A., A. S. Bradley, J. R. Waldbauer, R. E. Sum- mons, and E. F. Delong. (2007), PNAS 104, 5590-595. [2] Richard A. Krebs, Ulrike Alex- iev, Ranga Partha, Anne Marie DeVita, Mark S.Braiman. (2002) BMC Physiology- doi:10.1186/1472-6793-2-5. pncB[3]Berríos-Rivera, S. (2002) Metab Eng. (3):238-47. BBa_K1604010 and BBa_K731201 were expressed in NEB10β cells with retinal (10 uM) under anaero- bic conditions both in the light and in the dark. ATP levels were quantified with a luciferase based assay. PR shows a low ADP/ATP ratio to indicate more ATP production. We engineered E. coli to exploit sunlight and live better under the anaerobic conditions of Microbial Fuel Cell (MFC) and increase electrons production. OVERVIEW Proteorhodopsin (red) binds the retinal produced via the β-caro- tene (orange) and blh (yellow) pathway. Sunlight hits proteorho- dopsin, which produces an outward proton gradient. The ATP-syn- thase (green) is in this way activated. PncB (blue) increases NAD + intracellular concentration to help electrons production. araC-pBAD RBS pncB RBS araC-pBAD RBS PR RBS J23100 RBS blh araC-pBAD araC-pBAD RBS RBS β-carotene Riccardo Corbellari, Silvia Galvan, Elisa Godino, Claudio Oss Pegorar, Veronica Pinamonti, Alessandro Turcato, Moreno Zolfo The pigments were extracted by acetone and run on a HPLC re- verse phase column, which evi- denced the loss of β-carotene. Retinal was not detected sug- gesting that is being used in other metabolic pathways. 0 5 10 15 20 25 30 35 40 45 50 Time [min] β-carotene and renal soluon BBa_K1604020 BBa_K1604022 Absorbance retinal β-carotene 12 MFCs connected in series were able to power up a blue LED. The voltage was measured while changing an external resistance. The current was calculated by the Ohm law. BBa_K1604010 (red) showed higher voltage and current values compared to the negative control BBa_K731201, (blue). We were able to engineer an E.coli able to live in the absence of oxygen thanks to an increase of ATP in response to light activation of proteorhodopsin. We paved the road for retinal biosynthesis and built a new device able to produce more NAD + . Finally we constructed our own MFC proto- type and we powered up a blue LED. CONCLUSION and FUTURE PROSPECTIVE This is only one step forward: while we work to improve our ability to use bacteria to produce energy we all need to put effort to reduce our energy needs!
Transcript of Solar pMFC - 2015.igem.org2015.igem.org/files/poster/UNITN-Trento.pdf · 2015-10-08 · Team:...
Team: Alessadro Turcato, Claudio Oss Pegorar,Elisa Godino, Moreno Zolfo, Riccardo Corbellari,Silvia Galvan, Veronica Pinamonti.Special Thanks to:
Solar pMFCUNITN-Trento
A microbial fuel cellwith a light drivenE. coli engine
MORE ATP WITH PROTEORHODOPSIN.
0
5
10
15
20
BBa_K731201 BBa_K1604010
Dark DarkLight Light
araC-pBAD Proteorhodopsin
ADP/ATP ratio araC-pBADBBa_K731201
Proteorhodopsin BBa_K1604010
ADP/ATP ratio
Proteorhodopsin (PR)[1] creates a light-driven proton gradient, activating ATP synthase and increasing bacterial lifespan[2].
NEB10β expressing BBa_K1604010 (proteorhodopsin) were placed in the anaerobic anodic chamber of a small MFC togheter with chemical mediators (anode, blue methylene 100 uM; cath-ode ferricyanide 10 mM).
OUR BACTERIA ARE HAPPY IN THE MFC!WE HAVE MORE NAD+
pncB[3] catalyzes the rate limiting step of NAD+ biosynthesis. NAD+/NADH ratios were measured with a colorimetric assay, in an-aerobic conditions. BBa_K1604031 (pncb, yellow) and negative control BBa_K731201 (aracpBAD, green).
Our Solar pMFC under costruction (left) and at work (right). It is composed of 6 cathods in parallel that share the same anodic chamber. We obtained a maximum power of 109 μW with a cur-rent of 467 μA (external resistance of 500 Ω).
We succesfully produced β-carotene (B-Ba_K1604020) which is cleaved by blh in two molecules of retinal (BBa_K1604022) in NEB10b cells.
WHERE IS OUR RETINAL?
ATTRIBUTIONS:Instructors: Cristina del Bianco, Sheref S.Mansy, Olivier Jousson, Martin HanczycProteorhodopsin: S.G, R.C, E.G, blh; V.P, pncB: R.C, Mtr: A.T, MFC: A.T, R.C, Interlab: E.G, C.O.P, wiki master M.Z. Human practices: E.G. Prototype: A.T, R.C with the support of the Physicis Dept. workshop and Damiano Avi, Mariano di Mauro and Gigi Minati for helpful suggestions. Carolyn Ajo Franklin for bacteria with Mtr system. Martin Hanczyc for providing MFC materials. Graziano Guella for access to the HPLC. Gian Marco Franceschini for helping with the logo and graphics. The Mansy lab for some help in interlab study.REFERENCE:Proteorhodopsin and blh [1] Martinez, A., A. S. Bradley, J. R. Waldbauer, R. E. Sum-mons, and E. F. Delong. (2007), PNAS 104, 5590-595. [2] Richard A. Krebs, Ulrike Alex-iev, Ranga Partha, Anne Marie DeVita, Mark S.Braiman. (2002) BMC Physiology-doi:10.1186/1472-6793-2-5. pncB[3]Berríos-Rivera, S. (2002) Metab Eng. (3):238-47.
BBa_K1604010 and BBa_K731201 were expressed in NEB10β cells with retinal (10 uM) under anaero-bic conditions both in the light and in the dark.ATP levels were quanti�ed with a luciferase based assay. PR shows a low ADP/ATP ratio to indicate more ATP production.
We engineered E. coli to exploit sunlight and live better under the anaerobic conditions of Microbial Fuel Cell (MFC) and increase electrons production.
OVERVIEW
Proteorhodopsin (red) binds the retinal produced via the β-caro-tene (orange) and blh (yellow) pathway. Sunlight hits proteorho-dopsin, which produces an outward proton gradient. The ATP-syn-thase (green) is in this way activated. PncB (blue) increases NAD+
intracellular concentration to help electrons production.
araC-pBAD RBS pncB
araC-pBAD RBS PRaraC-pBAD RBS PR
RBS PRJ23100 RBS blh araC-pBADaraC-pBAD RBSRBS β-carotene
Riccardo Corbellari, Silvia Galvan, Elisa Godino, Claudio Oss Pegorar, Veronica Pinamonti, Alessandro Turcato, Moreno Zolfo
The pigments were extracted by acetone and run on a HPLC re-verse phase column, which evi-denced the loss of β-carotene. Retinal was not detected sug-gesting that is being used in other metabolic pathways.
0
5
10
15
20
25
30
35
40
45
0 5 10 15 20 25 30 35 40 45 50
Time [min]
β-carotene and retinal solutionBBa_K1604020BBa_K1604022
Abso
rban
ce
retinal β-carotene
12 MFCs connected in series were able to power up a blue
LED.
The voltage was measured while changing an external resistance.The current was calculated by the Ohm law. BBa_K1604010 (red) showed higher voltage and current values compared to the negative control BBa_K731201, (blue).
We were able to engineer an E.coli able to live in the absence of oxygen thanks to an increase of ATP in response to light activation of proteorhodopsin. We paved the road for retinal biosynthesis and built a new device able to produce more NAD+. Finally we constructed our own MFC proto-type and we powered up a blue LED.
CONCLUSION and FUTURE PROSPECTIVE
This is only one step forward: while we work to improve our ability to use bacteria to produce energy we all need to put e�ort to reduce our energy needs!
![ﻲﺷزﻮﻣآ ﻪﭽﳜرﺎﺗ ﺪﻬﺸﻣ ١۶/٨٩ لﺪﻌﻣ ﺎﺑ ١٣٧٢ ...full)[1].pdfNoemani H, Fakhar H. Detection of Chlamydia trachomatis by PCR – ELISA in infertile](https://static.fdocuments.pl/doc/165x107/5f4f50082afa395c63034459/iiii-iiioeii-iiii-iioei-ii-.jpg)
