Uppsala University, Sweden Department of Photochemistry and Molecular Science. Hydrogenproduction with Cyanobacteria

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2 Uppsala University, Sweden Department of Photochemistry and Molecular Science Hydrogenproduction with Cyanobacteria

3 Supervisors and Instructors Wetlab: Dr. Thorsten Heidorn Daniel Camsund, PhD student Modelling: Mats Walden, PhD student Institute for Cell and Molecular Biology at Uppsala University

4 The Uppsala igem Team Wetlab: Anders Kristoffersson (Sweden) Erik Florman (Sweden) Karl Brune (Germany) Ruiqing Ni (China) Modelling: Jonatan Halvardson (Sweden)

5 Nowadays Problems The world has a lot of problems: Climate Change Water shortage Food shortage Energy To be continued.. Beer in Sweden is incredibly expensive..

6 Booze Bugs The aim of our project Produce alcohols, in particular ethanol & isobutanol. Directly from sunlight With cyanobacteria Investigate ways to increase production of alcohols of Synechocystis sp PCC 6803

7 The Future Imagine if park areas could look like this and produce green energy!? PhotoBioReactor designed by Charles Lee

8 The issue of ethanol from agriculture Today 25% of the US grain harvest is used as fuel. 1 1 Scientific American May 2009, Could food shortages bring down civilization. Lester R Brown

9 The issue of ethanol from agriculture 175 million people in India is living on food produced from fossil water. 1 >1 Billion people is hungry in the world today Hungry People in the world today, Source: UN 1 Scientific American May 2009, Could food shortages bring down civilization. Lester R Brown

10 The issue of ethanol from agriculture The 25 % of U.S. grain harvest devoted to fuel, could potentially feed half a billion people. 1 This leads to the conclusion that agriculture based fuels can not substitute fossil fuels on a broad basis. 1 Scientific American May 2009, Could food shortages bring down civilization. Lester R Brown

11 The issue of ethanol from agriculture The 25 % of U.S. grain harvest devoted to fuel, could potentially feed half a billion people. 1 So, what to do?! 1 Scientific American May 2009, Could food shortages bring down civilization. Lester R Brown

12 Non-fermentative Algae Fuel Production: Booze Bugs Potentially non-competitive with food production. Deployable at locations not suitable for conventional agriculture. Low usage of fresh water. Adaption to use salt water possible

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14 The ethanol project Introducing genes for ethanol production. ADH2 & Pdc (Zymomonas mobilis) Redirecting flow of substrate. Inhibiting the PDC complex, using an antisense RNA and a protein approach.

15 The Implementation Ethanol Constructs

16 Increasing substrate levels Inhibiting the PDC, pyruvate dehydrogenase complex Antisense RNA A set of RNA s complementary to the PDC subunit mrna s. Aiming to disturb translation of the PDC subunits. PirAB Pirin like proteins, shown to inhibit the PDC 1, and inducing fermentative pathways 2. Aiming to disturb functionality of the PDC. 1 A cyanobacterial gene encoding an ortholog of Pirin is induced under stress conditions. Yukako Hiharaa, Masayuki Muramatsua, Kinu Nakamuraa and Kintake Sonoikeb, FEBS Letters 574 (2004) Pirin Regulates Pyruvate Catabolism by Interacting with the Pyruvate Dehydrogenase E1 Subunit and Modulating Pyruvate Dehydrogenase Activity. Po-Chi Lai et al. Journal of Bacteriology, January 2007, p , Vol. 189, No. 1

17 The Implementation Inhibiting the PDC

18 Why Ethanol & Butanol Ethanol + Widely used as a fuel and chemical + Wt Synechocystis already quite tolerant against EtOH Require infrastructure investments Corrosive More hazardous compared to gasoline Low energy density Butanol + Equivalent to standard gasoline. Plug & Play More toxic to Synechocystis compared to ethanol Not a proven concept.

19 The Butanol project Existing pathways in the Registry Alberta igem Team 2007: Fermentative pathway for butanol production But: Fermentative pathway not optimal in aerobic conditions. Pathway shown by Shota Atsumi, Taizo Hanai, James C. Liao Non-fermentative pathways for synthesis of branchedchain higher alcohols as biofuels (Nature Vol January 2008)

20 The Butanol project Introduction of genes for isobutanol production, kivd (lactococcus lactis) and ADH2 (bakers yeast).

21 The Implementation Isobutanol Construct

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23 Modeling We want to describe the system without knowing the enzyme concentration e 0 or the death term k t. Assuming quasi steady state and that the system can be described as a one step reaction.

24 Modeling Setting up equations With some additional assumptions and some math..

25 Modeling Leads to an expression, that allows us to optimize the system in respect to the promoter activities. For a complete deduction please visit our igem wiki.

26 Problems Getting our hands on the kivd gene Trials with PCR on filmjölk, swedish yoghurt. Lots of mails to different people! Bingo! Bioneer synthesizes the gene

27 Problems PirA and the PirAB construct Problems with PCR for PirA Problems with assembly of the PirAB construct And due to lack of time this approach was abandoned.

28 Problems Synechocystis Not the standard lab bug Require special parts Doubling time about11h 3 weeks from transformation to measurements

29 Results Evaluation in E.coli (constructs with pllac) Why use a GC when we can play around with statistics? Construction of a PLS model using spectrophotometric data. Measure ethanol/butanol concentration using the PLS model.

30 Results

31 Results Clear systematic difference between functional and control constructs.

32 Results Clear systematic difference between functional and control constructs.

33 Results Clear systematic difference between functional and control constructs.

34 Results Evaluation of antisense RNA in synechocystis Synechocystis culutres carrying two of the antisense RNA constructs grows very slow (A1, B2), even in copper free medium. The other nine show inconclusive growth rates for copper induced versus non induced.

35 Future Prospects Algae bio fuels = Big business > 1 billion USD invested since 2007 Synthetic Genomics, A Craig Venter business backed by ExxonMobil Sapphire Energy Backed by Bill Gates Gold Rush for algae; Nature 461, (2009)

36 Tack o Hej, leverpastej Thank you for listening