Integrated Human Practice
When designing our project and analyzing relevant literature we found out that a significant part of feed production costs is spent on raw materials – meat and bone meal in particular, about one third of which is contaminated with fungus cultures producing toxic substances. Therefore, we decided to choose a yeast strain that could recover such damaged raw materials. Yarrowia lipolytica has become the right choice for solving this problem as these yeasts can feed on nonpolar carbohydrates among which are fungal toxins (for example, fumonisin).
When we started our work with yeast cultures we noted that our phytase was not getting more stable. After analyzing every possible reason for the failure and consulting with an expert in this field (Elena Issakova, senior researcher of the Federal Research Center for Fundamentals of Biotechnology of the Russian Academy of Sciences) who had faced the same problem in her practice), our team concluded that this protein was allogenic in yeasts and could be destroyed by yeast enzymes (mainly on its N-end). Our solution was to create a structure containing our phytase and a domain that would protect the protein from being destroyed in a yeast cell. As a result we created a construct containing our phytase and a cellulose-binding domain attached to its N-end. We entered this construct into a yeast cell and demonstrated that activity of our phytase increased. Thus, the detailed study of various aspects of our project influenced its design at two key stages: the developing of a new genetic structure and our choice of a basic strain of microorganism producers for expression of the construct created.
Our sponsor BIOCAD is one of the biggest international innovative biotech companies in Russia combining world-class research centers, advanced pharmaceutical and biological production, and pre-clinical and clinical trials meeting best international standards. The company develops and produces advanced classes of drugs for most complex diseases, such as cancer, HIV, hepatitis, etc., and is the only producer of drugs based on monoclonal antibodies in Russia and Eastern Europe. The average age of its R&D personnel is 28 years; therefore, it invests heavily in young generation. The company supports a number of local and national competitions, but had never heard of iGEM before we invited them to become our sponsor.
Our first meeting was when its representatives came from Saint Petersburg to Moscow to meet us and learn more about the competition, our team and our project. Actually, the meeting was a kind of our first trial to present our project and our guests were a kind of our first judges. Their question and comments helped us to understand our week points and correct what required correction.
First of all, they drew our attention to the problem which we had initially underestimated. In the very beginning of planning our work we believed that our major objective was to solve the problem of thermal degradation during granulated compound feed production. But our guests from BIOCAD advised us to pay the same attention to the problem of its degradation in animals’ gastrointestinal tract which is not less important. The problem of phytase degradation during feed production could be in theory solved through adding it to feeds after granulation, while protecting it from GIT acidic conditions requires other methods. This meant that our final product should meet two major requirements equally: surviving thermal degradation during feed production and surviving GIT conditions in animals. ‘Two-in-one’, so to say. And these factors finally determined our decision to ‘encapsulate’ phytase in yeasts.
Besides, we discussed with BIOCAD the prospects for practical implementation of our project and their advice was to go deeper into the problem of phosphorus deficiency in animals’ organisms to understand its consequences. To assess the contribution that our final product could make to breeding strong and healthy livestock, we had to analyze the problem more profoundly. Following BIOCAD’s advice we decided to reach out to hog farmers for further feedback on our project.
Later the company invited us to visit them in Saint Petersburg where the head of the molecular genetics lab gave us a lecture on gene engineering, schemes for obtaining recombinant proteins, DNA, classic cloning schemes, major methods for using PCR, transcription, translation, expression, etc. Besides, we had a tour around the company’s premises and visited its labs where company’s representatives told us about BIOCAD’s current developments and achievements. And of course we were impressed by the excursion to their monoclonal antibody substances production site.
The National Union of Hog Farmers
The best way to assess the demand for a product as well as all its pros and cons is to ask its end users directly. In our case the end users are farmers – not pigs of course. To have a better understanding of the problem of phosphorus deficiency in animals and the pitfalls that the project can face on the way to its real life implementation we sought for advice of the biggest national association of hog farmers – the National Union of Hog Farmers.
First of all, we needed more information on the role this macroelement played in growth and development of livestock animals. Vladislav Ovcherenko (General Director of the Ufa Center for Selection and Crossbreeding) gave us a detailed explanation:
The significance of minerals in feeding farm animals, their quantitative and qualitative content and the balance between individual substances is really tremendous. This is explained by the fact that these substances are quite literally found as basic cell components in every live organism. About 80% of phosphorus found in a live organism is concentrated in its skeletal system; the rest of it is distributed in other tissues. Phosphorus is indispensably involved in almost all metabolic processes. In particular, it is a component of adenosine triphosphoric acid. And ATP is the main source and transmitter of energy in organisms. Carbohydrate metabolism in animals is impossible without phosphorus which accelerates glucose absorption and breakdown. Along with that, phosphorus participates in lipid metabolis; its compounds facilitate choline production in an organism. Besides, phosphorus is an important stimulator and catalyzer of absorption of nutrients from feeds. The main role in phosphorus transformations in animals’ bodies is played by liver and gall. Therefore in cases of liver and gall disorders there can be problems with adsorption of such an important element even when the amount of it in a diet is sufficient.
We also asked the farmers about the estimated cost efficiency of using phytase instead of phosphorus supplements, the problem of manure disposal in Russia in terms of its environmental impact on soil and water, the problem of reutilization of raw materials contaminated with fungi, the prospects for and expected difficulties of practical implementation of our project, and its possible week points that would need further elaboration. Our conclusions based on their feedback are as follows:
Insufficient concentration of phosphorus in farm animals and poultry’s diets makes it necessary to add mineral supplements containing a lot of it to their feeds. Solving the problem of releasing phosphorus and improving its absorption from organic feed components is of great significance. Today in Russia enzymic complexes produced by foreign manufacturers are used widely. There are no national manufacturers in Russia producing phytase of high quality and activity and universal for any raw materials. Developing Russian technologies in this field is of particular importance in the context of the national import substitution strategy.
The economic benefit of using phytase will be higher when producing feeds for young and lactating animals as this period of development requires higher concentration of phosphorus in feeds. The product proposed in the project is necessary and has high potential for implementation. But when developing and finalizing the product for industrial implementation the following aspects should be taken into consideration: 1) it should not be one universal product for various species and age-sex groups; different feed recipes should be developed for different groups; 2) phytase dosage should be calculated and balanced separately for two functions of the product described in the project: phytase activity and adsorption of toxins in raw materials. This means that our product has all the chances to be a commercial success – at least in the national market, but some further elaboration is required in order to make it even more marketable.
Our customs experience
When it was time to clone our construct in the pSB1C3 vector that we had received from iGEM our team faced some difficulties: restrictions and amplifications failed and, when applied to agarose gel, DNA was not identified in any sample at all. Our detailed analysis showed that concentration of plasmids in the tube delivered was only 5 ng/mcL instead of 25 ng/mcL (the data were received on ThermoScientific NanoDrop 1000). According to our lab experts, such values were too low for any manipulations with the substance. As we were short of time to order plasmids additionally, we decided to amplify a plasmid from one of the BioBrick samples as a matrix. This measure turned out to be helpful and allowed us to build up the plasmid and make and submit our BioBricks in time. Pondering over possible reasons for this problem the team concluded that it could be improper storage of plasmids on their way to Moscow. The point is that the box sent by iGEM is marked ‘Do not freeze’ and more detailed instructions on which samples should be stored at room temperature and which of them – in a freezer are packed inside of the box that was not opened at the customs. Unfortunately, the customs procedures took quite long and as soon as we received the box we immediately placed all the reagents at proper temperatures but it was probably too late. These difficulties have not prevented us from completing our project in time but encouraged us to make the following proposal for the iGEM organizers: as the competing teams are located in many countries where delivery of distribution kits can take quite long it might be more rational and effective to send linearized plasmids meant for cloning in dried condition (same as BioBrick samples) in order to reduce the risk of damage to plasmids in case of delivery and customs delays.