Document:

Amendment to Contract

EXHIBIT 10.3

Amendment to Contract

Title: Root Transformation Gene Expression and Nematode Resistance Studies in Common Beans for Evolutionary Genomics

Principal Investigator:  Dr. Matthew W. Blair (Research Associate Professor) / TSU, Nashville, USA 

Visiting Scholar:  Dr. Xue Renfeng (Subdirector Legumes) / LAAS, Shenyang, China

Principal Institution:  Tennessee State University, Department of Agriculture and Natural Sciences, Lawson Hall, 3500 John A. Merritt Blvd. / Nashville TN

 Granting Entity:  Evolutionary Genomics / Lafayette, CO

Principal Grant Contact:  Dr. Walter Messier / Evolutionary Genomics Inc. 

Funding Source: Bill and Melinda Gates Foundation

Dates of ammendment:  May 1st 2014 to Sept 30thst 2015 with possibility for extension.

Budget for contract:  $100,000 USD plus 10% overhead as per Bill and Melinda Gates Foundation rules – total $110,000 USD for this request. 

Budget items:

				
	Item

	First year

	Second year

	Total

	Personnel

	24,000

	12,000

	36,000

	Hourly help

	11,000

	13,000

	24,000

	Greenhouse supplies

	3,000

	5,000

	8,000

	Lab supplies

	15,000

	12,000

	27,000

	Travel 

	3,000

	2,000

	5,000

	Subtotal

	56,000

	44,000

	100,000

	Overhead (10%)

	3,800

	3,200

	10,000

	Total

	41,800

	35,200

	110,000

Budget Notes:  Personnel:  full time of a visiting scholar for field and lab work paid at rate of $24K/yr; Hourly help:  will be contracted either through subgrant with Auburn Univ. or collaboration with Univ. of Tennessee, Ellington Ag. Station  who have been contacted; Greenhouse Supplies:  pots, soil substrate, fertilizer, herbicides, etc,  Travel: trips to Auburn or Alabama A&M for nematode work and advice, University of Kentucky for VIGS system training and potentially to Boulder or Seattle for meeting with sponsoring company and funding agency;  Overhead:  Bill and Melinda Gates Foundation regulations on overhead rate maximum of 10% for US based research efforts applied to this grant as TSU is American based and will be a sub-grantee of Evolutionary Genomics, Inc. where the same rate is being applied.

Ammendment for root transformation (70 thousand dollars)

1)

Overall Objective

·

The overall goal is to implement hairy root transformation in common bean (Phaseolus vulgaris L.) to test EG261-silenced and EG261 – overexpressed construct in bean tissues.  The hairy root system takes advantage of the greater potential of Agrobacterium rhizogenes (Ar) to infect bean roots than A. tumefaciens (At) to infect and transform meristematic shoots.  The other advantage is that in hairy root transformation there is no need for full plant regeneration through tissue culture or axillary bud meristems from germinating seed embryo axes like there is in At transformation.   

·

Given that EG261 is known to function against a root pest, the nematode Meloidogyne incognita, it is very appropriate to use Ar hairy root transformations to develop a testing system for the gene’s function.  The EG261 transformed hairy root systems can also be used to test for resistance to other root pathogens such as Fusarium spp. root rot and charcoal root rot caused by Macrophomina phaseolicola, both common pathogens in the USA and around the world.  Local isolates of both of these pathogens are being obtained at TSU.  Nematode screening can be conducted on campus as described in the next section.  Root rot diseases such as Fusarium root rot and charcoal root rot can be screened on site at Tennessee State University greenhouse with the help of the plant pathology program.

·

This work relates to the objectives 7c and 7d as described in the Evolutionary Genomics proposal scope of work first assigned in 2012 which is quoted here below:  

“ Task 7c Knockout and knock-in EG261 (July 31, 2014): Perform ‘knockout’ of EG261 ortholog in resistant varieties of beans. In parallel, ‘knock in' resistant alleles into sensitive varieties, in order to achieve gain-of-resistance phenotype. Finally, using line(s) in which the native EG261 ortholog has been silenced, knock in resistant alleles of EG261 to observe gain-of-resistance phenotype. 

Task 7d Knock-in common bean EG261 (July 31, 2014): Knock in soybean EG261 in order to determine if the transformed soybean version of EG2612 shows cross-species effectiveness.  

2)

Laboratory Work 

·

We have increased seed of a standard snap bean variety that can be used for Agrobacterium rhizogenes hairy root transformation.  The name of the variety is Bush Blue Lake (BBL) and is the most commonly grown Asgrow derived variety in the United States.  BBL is found in on-farm production and in home gardens. We currently have approximately 10 Kg of this seed grown from ten plots in a legume yield ceiling experiment grown in 2013.

·

Currently resistant and susceptible varieties of common beans exist for Fusarium root rot and for charcoal root rot but are not commonly available for nematodes, with all accessions being susceptible.  Therefore any resistance screening must take into account the need for a knock-in approach in common bean.  We suspect that overexpression of EG261 in the hairy root system will be a useful method of testing the gene.  Gene silencing or knock-out might be tried as a secondary method when resistant genotypes are confirmed for each of the pests and diseases described above.

·

Dr. Matthew Blair has recently been assigned a laboratory (Rm 131) for molecular biology and we have access to a common room for tissue culture (Rm 127).  We also have established an office for the visiting scholar Dr. Renfeng Xue and research assistants Ms. Abimbola Allison and Mr. Trevor Johnston in Rm 130 which is adjacent to the lab.  The lab is in the newly built Agricultural Biotechnology building with specifications and photos attached (Annex 1).  The full space available for the project is over 1,500 sq. ft.  An additional common area laboratory is available for DNA work with electrophoresis equipment and gel imaging systems.  In addition, an equivalent lab is across the hall for plant-microbe interactions which is where Dr. Margaret Mmbaga’s lab is located.  We are determining the amount of growth chamber space needed for our experiments and might need to order an incubator or develop a humidity chamber. 

·

The equipment for transformation is in place since we have two laminar flow hoods and one biosafety level II hood in shared space of the tissue culture room and also in the middle space between the labs of Dr. Matthew Blair and Dr. Suping Zhou.   Available in this same laboratory is an ABI Real time-PCR machine as well as regular Techne and Eppendorf PCR machines and common molecular biology equipment such as a Flask shaker, Ultraspeed Centrifuge, Table-top centrifuges, etc. Finally there is a Meta confocal microscope attached to an Axiovert 200M image capture system for analysis of the hairy roots and gene expression levels.  All other basic tools and equipment can be ordered including:  Paper towels, Aluminum foil, 250 ml Erlenmeyer flasks, Forceps, Pots, Petri dishes (100/15), Bent glass rod in a triangular shape, E-Tubes 1.5, 2.0, 5.0, Falcon Tubes 15, 50 ml, 10, 20, 200 and 100 ul tips, 3 ml syringe Needle (0.8 × 32 mm).

·

Dr. Matthew Blair has obtained A. rhizogenes K599 strain from collaborators Dr. Federico Sanchez and Carmen Quinto along with the methodology for hairy root transformation which is already published.   The transformation vector used by these colleagues was based on a moficiation of pCAMBIA1305.1 (Genbank: AF354045).  Therefore, we have also requested the entire set of publically available pCAMBIA vectors from Australia.

·

If needed, the EG261 gene can be tested for providing resistance to leaf diseases through a virus induced gene silencing system (VIGS) which Dr. Matthew Blair has requested from the developers of this technology in the University of Kentucky.  The plasmids needed for that work would be pGHopR1 plasmid (RNA1), pGRHanR1 plasmid (RNA1) and pGG7R2-V plasmid (RNA2).  Currently we are requesting APHIS permission for use of this Kentucky based bean virus in Tennessee but will require processing time.  The leaf pathogen Xanthamonas axonopodis pv. phaseoli has been isolated in Nashville from beans and is stored in the Plant Pathology laboratory so will make a good candidate disease to test when measuring the effect of EG261.

3)

Materials and Methods

·

The experimental procedure will be based on the method of Estrada-Navarrete et al. (2006) entitled “Agrobacterium rhizogenes Transformation of the Phaseolus spp. :  A tool for Functional Genomics” published in Molecular Plant Microbe Interactions.  The protocol for this work was further fleshed out in Estrada-Navarrete et al. (2007) with the tile “Fast, Efficient and reproducible gentetic transformation of Phaseolus spp. By Agrobacterium rhizogenes” published in Nature Protocols.

·

We will purchase all of the chemicals needed for the trials including 96% ethanol, 6% sodium hypochlorite solution, Solid LB medium, Liquid LB medium, Glycerol, Liquid PY medium, Peptone, Yeast Extract, Sodium Chloride, Agar, Boric acid, Calcium Chloride, Cobalt sulfate, Copper sulfate, Iron citrate, Magnesium sulfate, Manganese sulfate, Nalidixic acid/Sodium hydroxide, Potassium di-hydrogen phosphate, Potassium nitrate, Potassium sulfate, Sodium molybdate, Zinc sulfate.  We may need to buy 0.05% (v/v) plant preservative mixture (PPM) / sterile H2O (Plant cell Technology Inc.).  Bacteria and positive plants will be managed with Rifampicin/di-methyl-sulfoxide stock solution selection.

·

The potting of the BBL seed will be in small jiffy pots with vermiculite mixed with sterilized peat moss.  The seedlings (at five days after germination) will inoculated by syringe wounding in the hypocotyl below the cotyledonary nodes with 10 to 15 ul of a fresh culture of A. rhizogenes.  Transfer of plugs may be to larger pots with regular greenhouse potting mix.

N stock solution

Personnel, Equipment and Supplies:  

All of the above activities will be supported by Dr. Blair, who is full-time research associate profressor at TSU and Dr. Xue, the visiting scholar from China (Shenyang city in Liaoning province which is a major bean and legume production area near the coast of China Sea).  We can also obtain TSU graduate students and undergraduates to help with tissue culture and seedling growth.  A research assistant, Mr. Trevor Johnston from Nashville USA can transition from the field to the laboratory along with the first research assistant involved in cowpea phenotyping, Ms. Abimbola Allison who is from Nigeria.  Additional students have been or can be trained in plant breeding and plant pathology and will be mostly from sub-Saharan Africa, China or India, thanks to the established group of Dr. Margaret Mmbaga and Dr. Suping Zhou.   A research assistant will have been hired as of end of August.  The research assistant is from Nigeria and graduated from an MSc in Agricultural production specializing in biofuels and nutrient analysis (Ms. Abimbola Allison).  Gender balance will be considered in carrying out this project as will the potential for a multiplying effect from the trainees and researchers involved.  

Amendment for nematode testing (30 thousand dollars)

1)

Explanation of Objectives

·

The goal of this section is to test root systems of common bean (Phaseolus vulgaris L.) transformed with  EG261-silenced and EG261 – overexpressed constructs for resistance to root knot nematodes Meloidogyne incognita

·

The hairy root system takes advantage of the greater potential of Agrobacterium rhizogenes (Ar) to infect bean roots than A. tumefaciens (At) to infect and transform meristematic shoots.  The other advantage is that in hairy root transformation there is no need for full plant regeneration through tissue culture or axillary bud meristems from germinating seed embryo axes like there is in At transformation.   This ,ales the Ar system ideal for testing against nematodes.

·

Nematode screening can be conducted at Tennessee State University with nematode cultures provided from University of Tennessee / Tennessee Department of Agriculture Experiment station in Nashville.  Protocols for screening can follow those used for soybean in various laboratories in the US or Canada or those used for cowpeas in the University of California – Riverside.  

·

While we have not received any protocols per se from Univ. California, Evolutionary Genomics does have a close relationship with University of Wisconsin where the initial EG261 soybean constructs were screened for nematode resistance.

·

One important aspect of the work is that we will be using local strains of nematodes rather than risking the cross-state importation of strains especially from the intensive vegetable growing areas of the west.  A strain source was found by Dr. Steve Bosc (UT-Knoxville nematologist but stationed in Nashville) in community gardens sampled in Davidson country (greater metro area) and is being multiplied currently for a project to screen tomato in the TSU-AREC station in Cheatham county.  

·

If we fail to isolate enough nematodes for our work from the local Nahsville soils described above we have been offered strains of both root knot nematode and soybean cyst nematode from Auburn University nematologist Prof. Kathy Lawrence.  Auburn university is well known in the southeast for screening soil samples from the region and identifying nematode species and strains.  They have been collaborators before of various HBCU schools and Extension centers including Alabama A&M as well as Virginia State University.     

·

Finally, two other nematologists have been identified in the state of Tennessee, one at Univ. of Tenn.-Knoxville (Ernie Bernard) and one at Univ. of Tenn.-Martin (Heather Kelly Young).

2)

Laboratory Work 

·

From the above we can see that we have the contacts and strains almost in hand to begin the work of nematode screening should this part of the amendment to the project be approved.

·

Currently Steve Bost (UT-Elllington) is increasing root knot nematode strain from a local community vegetable garden in Nashville in his greenhouses on mineral soil.

·

This RKN nematode strain will be revived by growing in a 1:1 soil to sand mixture with Tomato and Okra which are very susceptible plants.

·

Once the nematodes are ready the soil can be frozen or used immediately to infect the Agrobacterium rhizogenes hairy root transformed plants. 

·

We will use the variety Bush Blue Lake (BBL) for all this work as it is the most commonly grown Asgrow derived variety of snap beans in the United States and in Tennessee especially where snap beans are more important than dry beans.  

·

The transformed hairy root BBL plants will be transferred from selective media to sterile soil in four inch diameter pots (useful for full expression of hairy roots).

·

We will infect the rhizospheres of the hairy root transformed BBL plants with approximately 2000 or 3000 adult nematodes from the nematode increase.

·

The infested plants of approximately three or four weeks of age (A. rhizogenes often delays development as more resources go to roots than to stems) will be placed in an incubator / growth chamber set for ideal above ground growth (18 0C night / 24 0C day)

·

Six weeks after infestation we will count the egg masses on the root system in the form of white cysts occupying cross-sections of the roots.

·

An alternative method of infestation is to isolate the nematode eggs which can be purified by the NaOCl method.  

·

Dr. Matthew Blair has obtained A. rhizogenes K599 strain from collaborators Dr. Federico Sanchez and Carmen Quinto along with the methodology for hairy root transformation which is already published.   The transformation vector used by these colleagues was based on a moficiation of pCAMBIA1305.1 (Genbank: AF354045).  

·

We have also requested the entire set of pCAMBIA vectors from Australia and are being guided to US labs that still carry the publically available vectors since they are now not being maintained in CAMBIA , Australia.

·

To start off with we are already inserting the EG261 gene into the transformation cassette with an adaptor approach that replaces the GFP protein of the original construct with this new gene of interest

·

When functioning the EG261 gene will run off of a 35S CaMV promoter in the hairy root system.

·

Growth chamber screening will also have the advantage of little movement of the transformed plants as the growth chambers are located within a pair of doors of the transformation hoods and in specialized facility inside the new Agricultural Biotechnology Building.  

Personnel, Equipment and Supplies:  

For the transformation work and testing of transformed root systems we will use the facilities of the new Agricultural Biotechnology building.  These include the laboratory assigned to Dr. Blair as well as adjacent lab collaboration with Dr. Zhou who has worked with transformed plants before.  Between the two labs is a specialized tissue culture room with two growth chambers, two bacterial shakers and two transfer hoods.  In terms of personnel, we have the same people as described with Dr. RenFeng Xue as our main researcher together with Dr. Matthew Blair plus access to the University of Tennessee and Department of Agriculture experiment station in Nashville (Ellington Station) where Dr. Steve Bost, a well-known nematologist is located.  

Research assistants Ms. Abimbola Allison and Mr. Trevor Johnston are based in the lab as well as the field but can help with the nematode screen.  A work study student can be hired for tissue culture work.  Currently, two work study students have been identified and one is a careful engineering student who would like a biological project to diversify his portfolio.  This student (Mr. Tarence Rice) is only a sophomore so he has plenty of time to carry out the project.  Nematode counts can be carried out with an optical microscope in the lab of Dr. Margaret Mmbaga and gene confirmation can be done if the green fluorescent protein tag is left with the EG261 gene constructs as the neighboring lab has a Meta confocal microscope attached to an Axiovert 200M image capture system for analysis of the hairy roots.  An ABI7300 qPCR machine is available for gene expression studies.Fee for Service Agreement

EXHIBIT 10.4

Fee for Service Agreement

This Agreement (the “Agreement”) by and between The Curators of the University of Missouri on behalf of the University of Missouri–Columbia with its principal offices at 310 Jesse Hall, Office of Sponsored Programs Administration, Columbia, Missouri 65211-1230 (“University”) and Evolutionary Genomics, Inc., with its principal offices at 1801 Sunset Place, Longmont CO 80501 (“Client”), is made under the following terms:

ARTICLE 01.  STATEMENT OF WORK

The University will undertake the Client’s project entitled “Development of transgenic soybean (Glycine max) with candidate genes for SCN resistance” under the direction of Dr. Zhanyuan J. Zhang, the College/School of Agriculture, Food, and Natural Resources, Division of Plant Sciences, at the University of Missouri–Columbia, substantially in accordance with the proposed program and toward the goals set forth in the service proposal (attached hereto as Attachment A and hereby made a part of this contract). The respective contributions of University and Client are described in Attachment A.  This Contract and Attachment A describes the expected statement of work. The term of this Contract begins when the University has received the plasmid constructs, DNA source materials, and other essential information on construct constructions from the Client (the “Start Date”). The statement of work may be amended prior to the Start Date.  If the University and Client cannot reach an agreement as to the amendment, then the Contract may be terminated by either party without any payment liability. Any change in the scope of work after the Start Date must be approved in writing by both the University and Client.  It is emphasized that this project is for research purposes only, i.e., testing the functions of the candidate genes for the proof of concept.  

ARTICLE 02.  PERIOD OF PERFORMANCE

The term of this Contract begins when the University has received the plasmid constructs, DNA source materials, and other essential information on construct constructions ("Client’s Materials") from Client (the "Start Date"), which Start Date will be confirmed by the University in writing (which may be by e-mail), and will continue through the date the University completes the tasks set forth on, and as indicated in the “time-table for milestone work” on Attachment A, unless otherwise amended or extended by mutual written agreement of the parties.

ARTICLE 03.  PROJECT COSTS/AWARD

It is agreed that the total project cost paid by the Client for this Contract shall be $213,020 unless changed by written amendment to this Contract.  The University’s budget for the period is set forth in Attachment A.

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ARTICLE 04.  INVOICE SUBMISSION AND PAYMENTS

Upon the Start Date of this Contract, the University will invoice Client for the installments of the total project cost in accordance with the milestones described in Attachment A.  All invoices will be paid within 30 days of the date received by Client, and the reports pertaining to this Contract will be sent to Client at:

Evolutionary Genomics, Inc.

1801 Sunset Place, Longmont CO 80501

ATTN:  Dr. Walter Messier

Phone: (303) 862-3222 Ext. 302

Fax:  (303) 862-3225

Within ninety (90) days of the end of the Contract period, the University will provide Client with a final statement of expenditures, if Client will request.

All payments for effort under this Contract will be made to The Curators of the University of Missouri. Checks will be sent to:

University of Missouri AR 

PO Box 807012

Kansas City, MO 64180-7012

ARTICLE 05.  TITLE TO EQUIPMENT

Title to all equipment, materials and supplies purchased under this Contract shall vest in the University at the time of acquisition of the items.

ARTICLE 06.  MATERIALS/RESTRICTIONS

Client shall provide to University certain materials, data and information proprietary to Client as specified in Attachment A (the “Materials”).  Upon receipt of the Materials, University will utilize its expertise and facilities to undertake the Project in accordance with the Research Plan.  University shall use the Materials solely for conducting the Project under this Contract and for no other purpose, including without limitation any commercial purpose or any research other than the Project.   University shall not sell, transfer, disclose or otherwise provide access to the Materials, any method or process relating thereto, any replicated forms, derivatives or descendants thereof, or any form of germplasm, plant or other materials containing the Materials (collectively “Plant Materials”), in whole or in part, or that resulted from the Project, or any material that could not have been made but for the foregoing to any person or entity without the prior written consent of the Client, except that University may allow access to the Materials to its employees, officers, and agents who require such access in order to conduct the Project and solely for purposes consistent with this Contract; provided that such employees, officers and agents are bound by agreement to retain and use the Materials in a manner that is 

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consistent with the terms of this Contract.  When the Project is completed, University will return any remaining Materials and Plant Materials to the Client, or otherwise dispose of the Materials and Plant Materials as mutually agreed by the Client and University.  University’s use of the Materials and Plant Materials and performance of its activities under this Contract (including, without limitation, conducting the Project) shall at all times be in compliance with all applicable laws, rules and regulations.

ARTICLE 07.  DELIVERABLES/REPORTS

The University shall provide written progress reports (no more than 2 pages) to Client periodically during the term of this Contract and a final detailed written progress report, which shall be submitted within ninety (90) days after completion or termination of the Contract, whichever occurs first.  University shall also make reasonable commercial efforts to be available via teleconference to discuss program as needed.

ARTICLE 08. RECORDS

The University shall maintain research records and accounts necessary to assure proper documentation of the project and accounting of all project funds.  The service experimental records shall be available to Client or any of its authorized representatives during the period of this Contract, and for three (3) years after completion or termination of the project, whichever is later.  In the event of audit or dispute, records will be retained until resolution thereof.

ARTICLE 09. TERMINATION

The term of this Contract begins on the Start Date and will continue until the earlier of (a) the completion of the project, (b) termination prior to Start Date as described in Article 01 or (c) termination in accordance with this Article. This Contract may be terminated with cause, by Client upon 30 days prior written notice to the other party. This Contract may be terminated by University only for Cause.  Cause for the University means the loss of key staff members and the unavailability of replacements. Upon receipt of notice of termination, the University shall make every effort to reduce or cancel outstanding commitments and shall incur no additional expenses. Client shall reimburse the University for expenses incurred up to the date of termination, including uncancellable obligations and reasonable termination costs, but in no event will such costs exceed the total project cost set forth in this Contract.  However, University shall reimburse to Client any amounts paid in advance by Client for work that was not performed as a result of such termination.  The extent of the parties’ reimbursement obligations will be based on the experimental records and accounts of project funds described in Article 07.

A party may terminate this Contract by the delivery of written notice to the other party if the other party materially breaches this Contract and does not cure the breach, if curable, to the reasonable satisfaction of the non-breaching party no later than 10 days after the non-breaching party delivers the written breach notice to the breaching party.  Unless otherwise specified in the breach notice, or unless the non-breaching party has cured the breach, the termination is effective 11 days after the date of the non-breaching party's breach notice.

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Expiration or termination of this Contract shall not relieve the parties of any obligation accruing prior to such expiration or termination.  Article 8, 10, 11, 12, 13, 14, 18, and 19 shall survive any expiration or termination of this Contract.

ARTICLE 10. PUBLICATION

The University shall not publish or otherwise disclose the results of any research performed under this Contract without providing Client sixty days for the opportunity to give written review and comment.  Each of Dr. Zhang, his key staff and the University, reserve the right to co-publish the results of this research project with Client, if he and/or his key staff makes important intellectual contributions to this project and only after written review and comment from Client.  Any comments relating to the co-authorship shall be in writing. University shall consider in good faith any comments thereto provided by Client and shall comply with Client’s request to remove any and all Client Confidential Information from the proposed publication/presentation and if information about any Invention is requested to be removed, that shall only be until such time as protection for the Invention has been sought.

ARTICLE 11. PATENTS AND COPYRIGHTS/INVENTIONS AND RESULTS

(a) It is expressly agreed that neither Client nor the University transfers by operation under this Contract to the other party any patent rights, copyrights, or other proprietary rights either party owns or controls as of the Effective Date of this Contract.  University acknowledges and agrees that Client has, prior to the date of this Contract, identified the genes that are the subject of this Project prior to entering into this Contract.  Such genes shall constitute Materials for purposes of this Contract.  Client shall own and retain all right, title and interest in and to the Materials. Materials, Results (defined below) and Material Inventions (defined below) shall constitute Confidential Information of Client.  

(b)  All ideas, inventions, techniques and other technology, whether or not patentable, and all associated intellectual property rights, that are created, generated, developed or discovered as a result of the performance of activities under this Contract shall be deemed “Inventions.”  Inventorship shall be determined in accordance with U.S. patent laws.  

(c)  Client shall solely own any and all Inventions that (i) are improvements, modifications, derivatives or enhancements to the Materials, and/or (ii) incorporate, utilize or rely upon the Materials, and result from University’s activities under the Contract whether alone or jointly by employees or agents of both University and Client (“Material Inventions”).  University hereby agrees to assign all right, title and interest in and to Material Inventions to Client. 

(d)  Other than Material Inventions, (i) each party shall solely own any Inventions created solely by its employees, agents, contractors or affiliates (“Sole Inventions”), and (ii) the parties shall jointly own any Inventions that are made jointly by employees, agents, or independent contractors of one party together with employees, agents, contractors or affiliates of the other party (the “Joint Inventions”). Except to the extent University is restricted by the license and option granted to Client under this Contract, each party shall be entitled to practice, license, assign and otherwise exploit the Joint Inventions without the duty of accounting or seeking consent from the other party.    

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(e)  University hereby grants to Client (i) a worldwide, royalty-free, irrevocable, fully paid-up, non-exclusive license to University’s Sole Inventions, and all intellectual property rights therein, for internal research and development purposes, and (ii) a six (6) month exclusive option period (to begin from Client’s receipt of the written Results or written disclosure of Sole Inventions and/or Joint Inventions, whichever occurs last) to obtain an exclusive, worldwide, royalty-bearing license to University’s Sole Inventions and University’s interest in Joint Inventions for research, development, manufacturing and commercial purposes for any and all fields of use (as determined by Client). Should Client exercise such option within this six (6) month period, a reasonable and customary royalty rate will be negotiated together with the other terms and conditions of the exclusive license. Client acknowledges that University’s Sole Inventions and Joint Inventions may be subject to rights of the U.S. Government pursuant to Title 35 Sections 200-204 of the United States Code and regulations promulgated thereunder, as applicable.

(f)  All data, results and information generated from or resulting from use of the Materials hereunder and/or performance of the Project (the “Results”) shall be owned by Client and University hereby assigns all of its, and its employees’, agents’, contractors’ and affiliates’ right, title and interest in and to such Results to Client.  

(g) University shall promptly disclose to the Client all Results and Inventions. University also shall ensure that any employees, agents, contractors and affiliates of University that receive the Materials as permitted above or otherwise participate in the Project hereunder agree to assign, and assign, to University all Results and Inventions made or generated by such employees, agents, contractors or affiliates.

ARTICLE 12. CONFIDENTIAL INFORMATION

During the term of this Contract and for a period of six (6) years thereafter, the University and Client (each a “Recipient”) shall use their best efforts to protect the confidentiality of, and keep confidential, all confidential and proprietary information provided by the other party (the “Disclosing Party”) hereunder (whether in written, oral or other tangible form, and identified in writing as confidential and proprietary or a person would reasonably understand to be confidential in nature (whether or not marked as such), and any Invention that is developed as a result of the Project conducted under this Contract (collectively the foregoing information with respect to a Disclosing party, “Confidential Information”).  The Recipient (i) shall not disclose to any third party, or use, such Confidential Information except as permitted under this Contract, (iii) shall use the Confidential Information of the Disclosing party only for the purposes expressly permitted by this Contract, and (iii) without limiting the generality of the foregoing, shall not use the Confidential Information of the Disclosing Party for the research, development or commercialization of products.

This obligation of confidentiality shall not apply to information which (a) is or becomes known publicly through no fault of the other party; (b) is obtained or learned by the receiving party from a third party entitled to disclose it free of a duty of confidentiality; (c) is already known to the receiving party free of a duty of confidentiality at the time of disclosure, as shown by the receiving party’s prior written records; or (d) is independently developed by the receiving party without access to or use of Disclosing party’s Confidential Information provided hereunder.

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Confidential Information of the Disclosing party may be disclosed by the Recipient to employees, agents or consultants of the Recipient, but only to the extent required to accomplish the purposes of this Contract. The Recipient shall use at least the same standard of care as it uses to protect proprietary or confidential information of its own to ensure that such employees, agents and consultants do not disclose or make unauthorized use of the Disclosing party’s Confidential Information.

The Recipient shall be permitted to disclose the Disclosing party’s Confidential Information solely to the extent that such disclosure is required by law or by order of any court or governmental authority, provided, however, that the Recipient shall first have given advance notice to the Disclosing party so as to permit the Disclosing party to attempt to obtain a protective order requiring that the Confidential Information so disclosed be used only for the purposes for which the order was issued or for such other legal requirement, and that the Recipient cooperates with the Disclosing party in such efforts. 

The Recipient agrees to return all copies and the original of any such Confidential Information upon expiration/termination of this Contract and/or the request of the Disclosing party, except that the Recipient may retain one (1) archival copy of such Confidential Information for the sole purpose of determining its obligations hereunder. 

ARTICLE 13. PUBLICITY / USE OF UNIVERSITY NAME

Client will not use directly or by implication the name of the University or the name of any member of the University’s technical staff working on this research project (other than information/data in deliverables, which may be disclosed by Client) for any product promotion or commercial publicity or advertising purposes, nor in any way the aims, policies, programs, products, or opinions of the Client without the prior written approval of the University; however, the Client or a third party may identify the University as the site where the research was conducted.

ARTICLE 14. NOTICES

All notices required by this Contract shall be made in writing and sent prepaid by certified mail.  For purposes of this Contract, the addresses of the parties are as follows:

University:

(Technical) Dr. Zhanyuan Zhang

Director and Research Associate Professor

University of Missouri–Columbia

1-33 Agriculture Building

Columbia MO, 65211-1230

Email: ZhangZh@missouri.edu

Phone:  573-882-9592

Fax:  573-882-1469

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(Business) Jamie Szabo, Financial Officer Office of Sponsored Programs Administration University of Missouri–Columbia

310 Jesse Hall

Columbia, Missouri 65211-1230

Email: grantsdc@missouri.edu

Phone: 573/882-7560

Client:

(Technical) Evolutionary Genomics, Inc.

1801 Sunset Place, Longmont CO  80501

ATTN: Walter Messier, Ph.D.

Phone: (303) 862-3222 Ext. 302

Fax:  (303) 862-3225

(Business) Steve Warnecke

Evolutionary Genomics, Inc.

1026 Anaconda Drive

Castle Rock, CO 80108

Email:  warnecke@comcast.net

Phone:  (303) 513-3510

ARTICLE 15. RELATIONSHIP OF PARTIES

The relationship of the University to Client shall be that of an independent Facility and nothing contained in this Contract shall be construed to create the appearance of an employer/employee relationship.  The University shall have no authority to represent itself as an agent of Client or to bind Client for any obligation or expense not specifically stated in this Contract.

ARTICLE 16. ASSIGNMENT

Except as provided below, this Contract shall not be assigned by either party without the prior written approval of the other party, such consent not to be unreasonably withheld or delayed; provided, however, that Sponsor may assign this Contract without such consent to any of its affiliates, to any purchaser of all, or substantially all, of its assets to which this Contract relates, or to any successor corporation resulting from any merger, consolidation, share exchange, or other similar change of control transaction.  Any purported assignment of this Contract in contravention of this Article 16 shall be null and void. The parties’ rights and obligations under this Contract will bind and inure to the benefit of their respective successors, heirs, executors and administrators and permitted assigns.  

ARTICLE 17.  CONTRACT MODIFICATION

Any agreement to change the terms of this Contract (including any change to Attachment A) in any way shall be valid only if the change is made in writing and signed by the authorized representatives of the parties. No oral agreements or conversation with any officer or employee of either party shall affect or modify any of the terms and conditions of this Contract.

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ARTICLE 18. INDEMNIFICATION

Client shall indemnify, defend and hold harmless the University, its employees, officers and agents from any and all liability, loss, damage and expenses (including attorneys’ fees) they may suffer as a result of third party claims, demands, costs or judgments which may be made or instituted against any of them by reason of personal injury (including death) to any person or damage to property arising out of the negligence or willful malfeasance by Client. Any such liability, loss or damage resulting from negligence or willful malfeasance by the University, its employees, officers and agents is excluded from this agreement to indemnify, defend and hold harmless.

ARTICLE 19. APPLICABLE LAW

This Contract shall be governed by the laws of the State of Missouri. 

ARTICLE 20.  USE OF PURCHASE ORDER

Client hereby agrees that, should Client use a purchase order to fund this Contract, any terms and conditions contained in the purchase order shall be considered deleted and not applicable for purposes of this Contract.

ARTICLE 21. ENTIRE CONTRACT

This Contract and attachments hereto contain the entire agreement between the two parties. In the event of any conflict with the terms of Attachment A and the terms of the Contract, the terms of the Contract will control. This Contract may be executed in two or more counterparts, each of which shall be deemed an original, but all of which together shall constitute one and the same instrument.

ARTICLE 22.  DISCLOSURE

Client and the University understand that University will follow its Conflict of Interest procedure with respect to this agreement.  Client will have the opportunity to review any description of this project prior to conflict publication. 

FOR THE CURATORS OF THE

FOR THE CLIENT

UNIVERSITY OF MISSOURI

                                                                                

                                                                               

By: Karen M. Geren

By: Steve B. Warnecke

Title: Submissions Specialist, OSPA

Title: CEO

Date:                                                    

Date:                                                    

MU Project No. 00048381

8

Schedule A

Work Order 

Related to Fee for Service Agreement 

Between University of Missouri - Columbia and Evolutionary Genomics, Inc.

Effective Date of _November 1, 2014

NOTE:  In the event of any conflict with the terms of a Work Order and the terms of the Agreement, the terms of the Agreement will control.

SCOPE OF WORK / BUDGET

Title of the Service Project

Development of transgenic soybean (Glycine max) with candidate genes for SCN resistance

Overall goals:

To develop transgenic soybean events carrying genes of interest for soybean cyst nematode resistance analysis

The specific objectives of this project are to: 

Assemble transgene expression cassettes including promoter, gene, and terminator and clone them into the Plant Transformation Core Facility (hereafter call “Facility”) standard binary service vector, resulting in a total of 6 different constructs. 

Produce and identify 4 to 6 events (average 5) from reach of six constructs with at least 100 seeds per event, provided that the genes of interest do not have negative impact on plant transformation and regeneration and seed production as well as transgene inheritance.  

Analyze primary (T0) transgenic soybean plants using herbicide leaf-painting, Southern blot analysis and real-time PCR. 

Analyze progeny plants (T1) of primary transgenic plants using leaf-painting to determine transgene inheritance and estimate the number of transgene loci. Three lines of each T0 event that has passed the transgene will be analyzed by real-time PCR to confirm transgene expression. Then, 10 progeny plants of each event carrying transgene will be advanced to T2 seeds.

9

Analyze T2 plants from each of the 10 T1 lines to identify at least 1 homozygous line for each event using leaf-painting. This homozygous line will be advanced to T3 seeds. If an extra homozygous line could be obtained out of 10 T1 lines, it would be kept as a backup and advanced to T3 seeds.

In case the selectable marker gene, bar, is suppressed to such a level that progeny analysis by leaf painting would not be feasible, PCR would be used to determine the segregation to identify the T2 homozygous lines. 

Methods:

Construct designs: 

The Client will provide gene sequences of interest within a cloning vector will allow one step cloning of assembled cassette into the Facility’s binary vector. All genes of interest are listed in Appendix A. In case such one-step cloning won’t be possible and need either PCR-amplification or synthesis of genes of interest, the associated charge including labor and supplies will be applied to the Client second payment (additional to the payment for the Final Milestone), upon written request from the Facility.

Mobilization and confirmation of constructs:

The six constructs will be mobilized into Agrobacterium tumefaciens and verified for their integrity by plasmid rescue and restriction enzyme digest.  This stage is an important first step to warrant a successful transformation. 

Development of transgenic soybean events and transformation system:

After the integrity of these constructs has been confirmed within the Agrobacterium the Facility will start transformation experiments as outlined in the time-table for milestone work weekly until the expected number of experiments are started. Facility will deploy the constructs in an order according to Client priority rank (Appendix A), or the cloning outcome (first come first serve) to avoid service project delay. Facility will use soybean genotype “Magellan” as transformation material and will employ its most recent Agrobacterium tumefaciens-mediated soybean transformation system (Zhang et al., unpublished) to develop transgenic soybean events. This system was derived from the protocols described previously (Zhang et al., 1999, Plant Cell, Tissue, and Organ Culture 56:37-46; Ohloft et al., 2003, Planta 216:723 – 735; Zeng et al., 2004, Plant Cell Reports 22:478-482) and is now capable of delivery foreign genes into various soybean genotypes. The time frame for each independent experiment from the start to the T1 seed harvest is about 9 to 11 months, dependent on the plant growing seasons and the impact of the transgene on plant growth and development.   

10

Experimental design and deliverables:

A series of coordinated, consecutive transformation experiments will be conducted to generate the desired transgenic events. Based on Facility’s previous experience, an empty construct will be required side by side with the constructs carrying the genes of interest in some transformation experiments to help evaluate the impact of the genes of interest on SCN resistance, and/or plant transformation and regeneration process, as well as transgene inheritance. Thus, for each independent experiment, when desired, at least two constructs may be deployed side by side, of which one is empty control and the other 1 or 2 carry genes of interest.  Each independent experiment will start with 400 explants and multiple independent experiments will be started for the six constructs. Based on previous experience with the Facility binary construct backbone, this should generate sufficient number (4 to 6) of T0 primary transgenic events per construct successfully transferred to the greenhouse. We anticipate an average of at least 100 seeds per event. The time frames for the milestone work are listed below and require a good coordination between Client and the Facility. 

For herbicide screen leaf-painting, glufosinate solution (100mg/L or higher) will be applied to young, fully-expended leaves of young plants and results will be taken 5-7 days afterwards. For Southern blot analysis, young, fully-expended leaves of young plants will be sampled. CTAB genomic DNA extraction protocol or commercial kits will be used and genomic DNA will be cut with the restriction enzyme that will cut only once within the T-DNA, allowing to identify the number of transgene loci. Radiative P-32 will be used to label the probe to detect transgene insert.

For real-time PCR, primers will be designed based on the sequences provide by Evolutionary Genomics, Inc. Reference gene suitable for transcript analysis of various soybean tissues will be 60s/ELF1b or equally effective alternative reference gene.  

For progeny analysis to determine transgene inheritance, segregation ratio, and identify homozygous lines, at least 18 progeny plants from each parental line will be analyzed by herbicide screen. If no segregation in T2 plants of T1 parental lines is found, additional 80 T2 plants of each line will be analyzed by the leaf-painting to confirm the homozygocity. At least one homozygous T2 line per event will be advanced to T3 seeds for down-stream SCN assays by another lab. We anticipate average of at least 100 T3 seeds (lines) of each event will be available for SCN assays.  

Staff and Facility Resources

The Facility has the capacity to complete this project. For this project one post-doctoral associate who is skillful in molecular biology study including project needed molecular bench work, one full-time Research Specialist who specializes in soybean transformation and one greenhouse manager who specializes in greenhouse management, plant care, and progeny segregation analysis will contribute to this project to secure the success. Located in the Sears Plant Growth 

11

Facility greenhouse, the facility has spacious growth rooms, culture incubators, and greenhouse room that are specifically assigned for soybean and other crop tissue culture and greenhouse growth.

Time-table for transformation work (excluding cloning work and progeny analysis)

			
	 

	Earliest delivery

	Latest delivery

	Task Name

	in weeks

	in weeks

	Total time from transformation of soybean cot-node to T3 seeds

	80

	117

	Seed germination

	1

	1

	Co-cultivation

	1

	1

	Shoot induction  

	4

	4

	Shoot elongation

	6

	16

	Rooting

	3

	6

	Acclimatization  

	3

	4

	Greenhouse

	17

	24

	Harvest T1 seeds 

	1

	1

	T1 analysis and harvest T2 seeds

	22

	30

	T2 analysis and harvest T3 seeds 

	22

	30

	Delivery of T3 seeds to SCN team (Final Milestone)

	 
	 

It is difficult to predict how long it will take to complete cloning work at this time. However, if only one-step simple cloning would be required, it should be done within 3 months. 

Continued from the last step of the above table, it will take average 5 months from planting T1 seeds to harvesting T2 seeds. Then, 1-2 months “drying seeds” will be needed to secure T2 seed germination for progeny test. Next, it will require another average 5 months to obtain T3 seeds while T2 homozygous lines of T1 lines of each T0 events will be identified. In total, at least one year will be needed from planting T1 seeds to harvesting T3 seeds. 

12

Budget (milestone payments):

Client will pay Facility a total of US$213,020 for the Work Plan based on successful completion of the milestone. Upon full execution of this Agreement, the University on behalf of Facility will invoice Client  in accordance with the following schedule and for the stated amounts:

$127,812

(60% of total expenses) Upon the start date of the designated project or upon full execution of this Contract, whichever is later.

$85,208

(40%) Final Milestone: At least average 100 T3 seeds of no less than one homozygous line per event from each construct will be delivered (e.g., to Dr. Henry Nguyen’s lab).  When a large percent of primary transgenic events showed susceptible to leaf-painting, suggesting that gene of interest suppresses the bar gene, Southern blot data on T0 and PCR data on T1 showing the presence of the transgenes will be used as a valid confirmation evidence. The Final Milestone will still be met when the empty control construct shows normal transformation rate and inheritance whereas the transgene of interest adversely affects selectable marker gene bar expression, plant transformation and/or regeneration, causing unusual lower rates of positive T0 transgenic events. In this event, if Client would need Facility to re-do the transformation experiments to obtain expected number of events, Client will pay the Facility extra labor and supplies based on the budget proposed by the Facility. The budget calculation will be based on the cost of labor and supplies as well as indirect cost, instead of the event production.  

Payment will be made within 30 days of invoice.  

Within ninety (90) days of the end of the Contract period, Facility will provide Client with a final written report containing the following information pertaining to the conduct of this Work Order:

·

Protocols used for: vector mobilization in Agrobacterium, Agrobacterium preparation, seed germination and explant preparation, Agrobacterium infection and co-cultivation, shoot induction, shoot elongation and rooting, greenhouse growth conditions and seed collection.

·

Metrics collected for each construct; total cot-nodes treated, total explants lost to contamination, total regenerating explants, total rooted lines sent to the greenhouse, average seed set per plant and frequency of soybean escapes or chimeric plants.

In the event that the gene of interest does reduce the transgene inheritance, leading to the reduced transgenic T0 events that can pass the transgene to the progeny, Client will pay the amount of fund to the Facility according to the inheritance scenario table and associated payment amount below. Such inheritance problem can be determined in the progeny segregation analysis of T0 events. 

Transgene inheritance scenarios: 

				
	Number of SCN races to test

	3

	Average number of T0 events per construct passes genes to  T1 

	3

	4

	5

	Grand total

	$138,642

	$175832

	$213020

13

AGREED TO AND ACCEPTED BY:

University of Missouri - Columbia

Evolutionary Genomics, Inc.

By: ______________________________

By: ______________________________

Name: Karen M. Geren

Name: Steve B. Warnecke

      

Title: 

Authorized Signer,Grants and Contracts

Title:  CEO

Date: ______________________________

Date: ______________________________

[In Duplicate]

MU Project ID 00048381

14

Appendix 1: Constructs carrying genes for functional analysis

					
	Construct number

	 
	 
	 
	Target Trait(s)

	1

	EG261pesca

	 
	 
	SCN resistance

	2

	EG261tomen

	 
	 
	SCN resistance

	3

	EG19micro

	 
	 
	SCN resistance

	4

	EG19tomen

	 
	 
	SCN resistance

	5

	EG261pesca/EG19tomen

	 
	 
	SCN resistance

	6

	EG19synthetic

	 
	 
	SCN resistance

	7

	 
	 
	 
	 

	8

	 
	 
	 
	 

	9

	 
	 
	 
	 

15

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