M A N N A L A B S

Dr. Dr. Flaig Rűdiger & MA Lange-Flaig Irén

Markets

Food:billion-$ market

Fodder:billion-$ market

Fuels:billion-$ market

Eco:billion-$ market

One Product Fits All

Food:billion-$ market

Fodder:billion-$ market

Fuels:billion-$ market

Eco:billion-$ market

Transgenic PlantsTransgenic Plants

Our Technology

Food:billion-$ market

Fodder:billion-$ market

Fuels:billion-$ market

Eco:billion-$ market

«FURLAC»DE 102007053992.6

PCT/EP20008/009596

«HIMBEER»DE102008038937.4

«YOGI»DE102008064223.1

.

Spin-offs huge market

Transgenic PlantsTransgenic Plants

Problems To Be Solved➲ The global energy supply is critical. – Fossil fuel reserves (coal, oil, gas) are limited.

– Regenerative energies are not sufficiently available. – Nuclear energy is politically unacceptable.

➲ Biofuels present a viable alternative only if they do not endanger food production.

– Without fuel mechanized agriculture is impossible.

➲ Arable land is decreasing in quantity and quality. – Malnutrition and starvation still affect millions. – Excessive agriculture worsens things.

➲ Fresh water is increasingly scarce.➲ The world population is still growing. – Industrialization also increases per capita demands.

➲ ...that can provide both food/fodder and biofuel to satisfy the growing demands of a growing population,

➲ ...that does not require arable land to grow and hence does not interfere with conventional agriculture,

➲ ...that can be used for «ecorestoration» in the widest sense, including soil amelioration,

➲ ...that is tolerant to salt water and thus does not increase fresh water consumption,

➲ ...that ideally withstands a wide variety of environ-mental conditions.

Thus we need something...

The Markets (1)➲ Food and fodder in the context of our patents:

Any plants may be used Suitable for direct human consumption Suitable also for feeding cattle Increased growth without need for fertilizer Do not exhaust the soil; with suitable plants such as

duckweed or algae, no arable land is required at all Salt resistance and stress tolerance Thus, they can be used to achieve a sustainable increase

in global agricultural productivity The global population could be fed effectively using the

plans presented here Huge net profits from licensing fees for a globally used

product

The Markets (2)➲ Biofuel in the context of our patents:

Significantly higher productivity than conventional biofuel plants

No competition with food and fodder production: Hitherto unused areas can be used

With a suitable choice of plants such as the alga Hæmatococcus which is naturally rich in hydrocarbons (carotenes), refined fuels can be obtained more cheaply

Global energy demands could be satisfied using the plans presented here

Huge net profits from licensing fees for a globally used product

The Markets (3)➲ «Eco»: A wide variety of environment-related

applications are envisioned, including: CO

2 sequestration to combat climate change;

emission minimization in power plants Clearing of low-MW silicate from waste waters and

synthesis of phyllosilicates to improve soils Occupation of natural habitats to control pest organims

and their vectors (e. g. covering open water to suppress malaria mosquitoes)

Removal of oil and petrol contaminations from soil and water

Huge net profits from licensing fees for several globally used products

The Markets (4)➲ Other applications:

Please see the PCT/EP2008/009596 for details on what kind of possible applications has been claimed so far

Minor possible applications claimed therein include: – Production of paper and cardboard

– Expression of pharmaceutically valuable proteins – Production of sugars, «bioplastics» etc. – Pest control

Still further applications may be developed

Investment Safety➲ The project comprises a plurality of new techno-

logies («Furlac», «Himbeer», «Yogi», etc.).

➲ Each new technology can be put to a variety of uses (see «Spin-Offs and By-Products»).

➲ Most uses (e. g. food, energy) are of global importance and unlikely to be obviated by unforeseeable changes or advances.

➲ Even if some technologies and/or applications should fail to deliver their promises, there are still plenty of market opportunities to make the project an economic success.

Knight Moves

Soil amelioration

Waste water treatment

Pest control

Fuel Food and fodder

CO2 sequestration

Removal of oil slicks

Paper, sugar,

bioplastics

Core Project: «Furlac»

➲ Improve plant growth rates – – Increase utilization of sunlight

➲ Make plants independent of fertilizer – – Enable plants to use atmospheric nitrogen

➲ Render plants resistant to salt water – – Pump salt out of the cells

➲ Environment safety feature: Limit maximum growth –

– Built-in senescence

Core Project: «Furlac»

➲ Improve plant growth rates – Increase utilization of sunlight

➲ Make plants independent of fertilizer – Enable plants to use atmospheric nitrogen

➲ Render plants resistant to salt water – Pump salt out of the cells

➲ Safety feature: Limit maximum growth – Built-in senescence

Requirement for working effectively

Increasing Sunlight Utilization (1)

➲ plants use only 4% of incoming sunlight

➲ only red and blue light is absorbed in the chloroplasts, green light is reflected

➲ FRET (Foerster Resonance Energy Transfer): this physical effect allows fluorescent substances to directly transmit energy to one another

➲ a fluorescent substance in the chloroplasts absorbing green light could utilize the rich green fraction of sunlight by transmitting it to the chlorophyll

Increasing Sunlight Utilization (2)

➲ Nobel Prize 1999 (Blobel): How to place proteins at subcellular locations

– Any protein can now be placed in the chloroplasts

➲ Nobel Prize 2008 (Chalfie): Fluorescent proteins – several proteins absorb green to yellow light

and can transfer it to chlorophyll

➲ Taken together, these two discoveries basically solve the problem

➲ Subtleties covered by «YOGI technology»

Fertilizer Independence

➲ Enzymes utilizing atmospheric nitrogen are known, but they need lots of energy and are sensitive to oxygen

➲ This energy plus is provided by increased sun-light utilization

➲ The enzymes are «blobeled» to places in the cell where oxygen is scarce (mitochondria)

➲ Subtleties also covered by «YOGI technology»

Compatibility with salt water

➲ Enzymes pumping salt out of the cells are known (e. g. from the human kidney), but they need lots of energy.

➲ More energy is provided by increased sunlight utilization.

Built-in Senescence➲ In the absence of the enzyme

telomerase, cell growth is limited, and cells become senescent.

➲ Place therefore the cells' intrinsic telomerase under external control (e. g. with Bujard's TET system),

➲ and you get a double advantage: – No danger of biosphere overgrowth by the trans

genic plants

– (farmers) cannot illegally propagate the plants

What To Do?

➲ Part A: Construct chloroplast fluorescence gene in the chloroplast and place it in plant cell → energy

➲ Part B: Construct mitochondrial nitrogenase gene and place it in plant cell → nitrogen

➲ Part C: Provide salt pump gene and place it in plant cell! → compatibility with salt water

➲ Part D: Replace the plant's telomerase with regulated telomerase ! → safety

– Not required for proof-of-principle stage!

Units of the Project

➲ (I) – Core project: – comprises parts A, B, C and D – «YOGI technology» may be required

➲ (II) – Add-ons: – comprises parts E, F and G – «YOGI technology» mandatory

➲ (III) – Spin-offs and by-products: – interesting individual applications – further «extras» to be determined after consult- ation with sponsors and interested 3rd parties

Plants of Interest➲ We develop our processes by using unicellular,

microscopic model organisms which are nevertheless already useful in their own right:

– Chlorella – Hæmatococcus

➲ After proof-of-principle, the

results are transferred toduckweed

➲ or to any other plant ofinterest (including maize,rapeseed, soybean, etc.)

Dimensions of the Work (1)

➲ Each of the four parts is roughly comparable to a master's thesis.

– If it works, it works. No scale-up problems in biology. – In the best case, each part could be done in a month.

➲ Each of the four parts is basically simple but there may be sticky spots.

– We have anticipated this by designing the powerful «YOGI technology».

– However, its implementation will require work and expenses. – Rather allow for more conservative timelines.

– 10 months for each of A, B and C + 4 months initial setup – Proof–of–principle for A + B after 24 months

Dimensions of the Work (2)➲ Apart from our research work, in work with biological

systems there is generally seen very much routine labor:

– 60 – 70% of all work can be done by technicians: – Maintenance of cell cultures and plants – Production of buffers, media and other «commodities» – Verification of lab equipment

– 10 – 20% can be done by unskilled lab workers – Cleaning glassware and other equipment – Disposing of waste material – Other chores

➲ Sufficient staffing is indispensable: – for core project: at least + 3 technicians + 1 worker – preferably: + 6 technicians + 2 workers, however, – the more, the better! (Swifter development saves time,

resulting in better return-on-investment.)

Dimensions of the Work (3)➲ Our Patents:

– «Furlac» (PCT/EP2008/009596) – enters the national / regional phase in summer 2010:

– translations – dissolves into a bunch of national/regional applications – all PCT countries should be considered

– «Yogi» (DE 10 2008 062 965.0) – «Himbeer» (DE 10 2008 038 937.4)

➲ Trademarks, «sorten» and other IP: – to be filed – individual products to be protected later.

Timelines➲ Projected start: April 2010➲ Ready for business: August 2010 – starting with parts A+B – other parts (including «YOGI») concomitantly if

sufficient staff and funding are available.➲ 1st milestone: April 2012 – proof of principle for parts A+B – start parts C+D if not begun before – decision on «add-on» project parts.➲ 2nd milestone: April 2013 – core project completed, including P.o.P. for C+D – other parts depending on staff and funding – evaluation of progress in «add-on» parts

Spin-Offs & By-Products➲ Various applications of the plants – food, fodder, fuel as mentioned above – technical systems incorporating these plants: – waste water plants – zero-emission power plants

➲ Further applications of the technology: – e.g. the «YOGI» technology for the study of

molecular interactions: – commercially useful enzymes – therapeutics and diagnostics – rapid treatment of emergent diseases (Ebola and friends) – affordable personalized therapy (cancer) – with DE102007005191.5: drug delivery system – with DE102007005192.3: possibly treatment of in-

flammatory and degenerative diseases

How is it done?➲ Molecular biology lab work mostly consists of

iterations of the following cycle:

– Obtain DNA sequences – optionally: Modify them – Put them together – Verify them – Use bacteria to produce lots of them – Place them in the target organism – See what happens and evaluate the results – Modify and start from the beginning until the

desired results are reached

➲ This is the basis for workflows and equipment.

Obtaining DNA Sequences➲ Getting sequences from databases → computer & internet➲ Synthesis → to be outsourced➲ Getting DNA/RNA from organisms → no special equipment required➲ PCR (Polymerase Chain Reaction) → thermocycler, polymerase & friends➲ Antibody-coding sequences from hybridomas → to be outsorced➲ «YOGI» → liposome preparation, zetasizer, facsort

Modifying & Joining Sequences

➲ PCR – thermocycler

➲ Restriction («cutting») – restriction enzymes

➲ Ligation («pasting») – ligase

➲ Verification: by electrophoresis and sequencing – electrophoresis kit – sequencing to be outsourced – alternatively (high workload, e. g. in «YOGI»):

sequencer

DNA Mass Production➲ Bacterial culture

– incubators– shakers

➲ Preparation– Sorvall/Beckman centrifuge– benchtop centrifuge– Qiagen kits

➲ Verification– electrophoresis and sequencing

Into the Target Organism

➲ Transfection of plants cells – electroporation (Huang et al. 2006) – alternatively: «gene gun» – Caution: May require firearms permit!

➲ Selection of genetically modified cells – chemical selection – selection by fluorescence: facsort

➲ Additional characterization – fluorescence microscope with camera

Maintaining Target Organisms

➲ incubators and shakers with illumination

➲ light meter

➲ microscope with Neubauer chamber

➲ ideally: Coulter counter

➲ autoclave

Other Needful Things➲ Library access

➲ Cooperation with university/business for specialized analyses and syntheses

➲ Miscellaneous laboratory equipment – detailed list attached

➲ Garbage disposal – all biological matter can be sterilized and discarded

➲ Housing

Permits and Dangers

➲ Required operating permit: Biosafety S1 (lowest level)

➲ Project Leader permit (Dr. Dr. Flaig has it)

➲ Small amounts of lab chemicals

➲ No «immissions» or large-scale waste production, no explosives, no biohazard waste

➲ Others permits required? - Consult legal expert for details of the local situation!

Add-Ons➲ part E: silicate polymerisation – remove low-MW silicates from waste water

(thereby preventing diatom blossoms) – produce phyllosilicates to increase retention in

soils➲ part F: amphipilic peptides and oxidases – secrete detergent-like peptides and oxidating

enzymes – dissolve, oxidate and devour petrol contaminations➲ part G: «Himbeer» – isolate resistance determinants from tardigrades – create plants with extreme resistance to heat, cold

and drought

Glossary (1)

➲ PCR: – method to amplify and/or modify a piece of DNA – requires thermocycler and reagents➲ Thermocycler: – device for cyclic heating and cooling of samples

suitable for PCR - € 3000,-➲ Liposomes: – miscroscopic bubble-like structures used in YOGI – require LiposoFast or LIPEX device for manufacture

and zetasizer / facsort for measuring – prices upon request only (avestin@avestin.com)

Glossary (2)➲ Zetasizer: – device for determining size and surface charge of

liposomes or similar particles - € 2500,-➲ Coulter counter: – device for counting and sizing cells - € 4000,-

(Ebay: € 500,-)➲ Microscopes: – 2× inverted microscope for cell culture - 2× € 1500,- – Fluorescence microscope with camera - € 5000,-➲ Facsort: – device for measuring and sorting cells and liposom-

es by size and fluorescence - € 50'000,- with sorting, € 30'000,- without

Glossary (3)➲ Centrifuges: – required for all kinds of precipitations and pelleting come in two basic sizes: – benchtop centrifuge – 3× € 1500,- – large and cooled – 2× € 3000,-

➲ Electrophoresis: – sorting macromolecules by size – comes in two flavors, both comprising a large

number of small components: – for proteins – € 1000,- total – for DNA – € 1000,- total

➲ Autoclave: – to sterilize things - € 1000,-

Glossary (4)➲ Enzymes: molecular tools for anything – Polymerase: makes DNA

– Restriction enzymes: cut DNA– Ligase: pastes DNA

➲ Incubators and shakers: – required to keep microorganisms happy - € 3000,-

for comprehensive set– must be equipped with illumination – contact local craftsmen to install this

➲ Qiagen: – kit of materials to isolate DNA from bacteria

➲ Sequencer: – to get the sequence of DNA - € 5000,-

Glossary (5)

➲ Electroporation: – force DNA into cells to create transgenic

organisms by means of subjecting them to electric shock – Requires electroporator - € 2000,-

➲ Biolistic: – «gene gun» - alternative to elecroporation, shoots

micro-projectiles into the cells - € 8000,- – legal status ?

➲ Neubauer chamber: – add-on for microscope to count cells - € 50,-

➲ Furniture and miscellaneous instruments: – listed separately