Team:Stuttgart/Description

Project

Description

Abstract

During the evaluation of topics for our project, we browsed through various themes inside the field of synthetic biology. Two ideas caught the attention of the whole team. Therefore, we combined them resulting in one project with two challenges. PhyCoVi was brought to life. With this project, we equally contribute to the research of scientists by accelerating laboratory work in molecular biology and offer new phototrophic substrate sources for biotechnology, to reduce the usage of food-related nutrient sources. Firstly, we apply synthetic biology tools to support the protein biosynthesis of the already fast-growing host Vibrio natriegens. In this project we want to achieve this goal by increasing the cellular tRNA availability to fasten the translation. Our second goal aims to increase the sustainability of industrial biotechnology. We chose an approach that targets substrate production for the cultivation of microorganisms and does not interfere with food production. Therefore, we outline a way in which biomass is produced by carbon dioxide fixation and light. We want to use phototrophy, more precisely phototrophic algae, which are able to produce biomass from nothing but salty water, air and light (and time). The idea of "Creating proteins out of air and sun” combined both parts of PhyCoVi and lead us to generate a phytotropic codon-optimized Vibrio natriegens.

Vibrio natriegens

Introduction - Vibrio

Over 60 years ago Vibrio natriegens was firstly described as Beneckea or Pseudomonas natriegens and later sorted to the genus of Vibrio. Vibrio natriegens is a non-pathogenic marine organism found commonly in brackish coastal waters2. V. natriegens inherits one of the fastest growth rates for non-pathogenic bacteria and captured our attention because of the increasing interest in novel host-organisms for biotechnology1. with a generation time of less than 10 minutes, V. natriegens grows significantly faster than E. coli.2 An important feature of Vibrio natriegens is that his genome is encoded onto two chromosomes, leading to two replication sources that allow parallel replication. Furthermore, recent studies from 2018 reveal that V. natriegens have an estimated 115,000 ribosomes during the exponential phase compared to E. coli which can reach up to 90,000 per cell3. Parallel replication and the fact that V. natriegens possess more rRNA operons controlled by stronger promoters leads to an increased number of ribosomes compared to E. coli, which corresponds to the fact that rapid growth is limited to the ability of protein biosynthesis4. Thereby, Vibrio natriegens capacity to perform extraordinary well in protein biosynthesis in vivo and in vitro as well show its potential to become the new commonly host.


Besides the rRNA quantity of an organism, tRNA plays an important role.Studies show that rRNA respectively ribosomes are important for the translation initiation and tRNA for the elongation5. Thereby, both rRNA and tRNA are vital during protein biosynthesis. While initiation translation the 5’ mRNA sequence plays an important role, it is recognized by the ribosome which binds and starts the translation6. Earlier studies could show, that 30-40 nucleotides of the 5’ end of ORFs undergo evolutionary selection which favors a weak mRNA folding at the ORF’s 5’ end7. Studies could show that mutations in these regions can vary the protein translation efficiency. It is necessary to mention that another study8 couldn’t observe this behaviour with two reporter genes. Nevertheless, good translation initiation is crucial for a high translation efficiency so that more ribosomes can bind to the mRNA.

After the translation initiation, the elongation rate is a promising way to fine-tune a translation to the optimum. Essential for a high elongation rate is the tRNA availability of the used codons within the mRNA sequence. The codon usage describes the frequency with which individual codons are used inside the genetic code. This codon usage varies from organism to organism and even on the genetical level inside the genome of each organism. Previous studies5 investigated codon usage in terms of translation rates for commonly used codons. Good expression requires a high translation initiation rate, but the key to optimal expression is high translation elongation9. When it comes to the expression of heterologous genes, codon bias is often seen as one of several potential bottlenecks leading to poor levels of expression or the formation of inclusion bodies. This is due to the poor tRNA availability of rare codons during translation of mRNA, which reduces the translational elongation rate. Therefore, we want to increase the cellular availability of rare tRNAs to increase the expression of heterologous genes with an increased ratio of rare V. natriegens codons.

Time is a key factor in every part of life and work. Synthetic biology has always been interested in engineering e.g. the growth rate of strains by e. g. reducing the genome of a host10,11. On the other hand, these genome editing approaches can result in a less tolerant or productive strain compared to the wildtype. Therefore, our approach with Vibrio natriegens shows an alternative by using a host with an already high growth rate to focus strain engineering on other important issues, such as the use of non-natural substrates for sustainable industrial biotechnology.

After this brief introduction about Vibrio natriegens and protein biosynthesis we now going to tell you how we planned to contribute to establish Vibrio natriegens for further applications during this competition.

In silico / dry lab

As already described, the availability of tRNAs can be a key factor in fine tune the translation elongation when it comes to optimising protein translation. We used in silico tools to identify codon usage and tRNAs of the new host Vibrio natriegens and outlined the topic.


Codon usage and tRNAs

>Firstly, we generated the codon usage table applying the High-performance Integrated Virtual Environment-Codon Usage Tables (HIVE-CUTs) tool on the coding sequences for Vibrio natriegens (DSM759). After the rare codons were identified, a tRNA-scan12 was performed throughout the genome of Vibrio natriegens to find the corresponding tRNA genes. This is critical due to the differentiation of cellular aminoacyl tRNA synthetase (AaRS), which recognizes the unloaded tRNA at a specific location within the tRNA's structure and only accepts and loads if the tRNA is correctly recognized.



Table 1: Identified rare codons used in Vibrio natriegens and found with the tRNA-Scan tool12 .
Codon Frequency
AGG (1.42 codons/1000 codons)
CGG (1.52 codons/1000 codons)
CCC (2.93 codons/1000 codons)
TGC (3.56 codons/1000 codons)
AGA (4.54 codons/1000 codons)
TGC (5.17 codons/1000 codons)


GeCoS – GenCodonSearch Tool

To get a better overview of the codons used for a gene or to identify codons that contain a high number of rare codons, we offer an algorithm written in Python3 so that everyone can use it freely and easily. GeCoS has enabled us to quickly identify and characterize genes that may not be expressed due to the codons used. To use this tool, you simply need to know which codons you are looking for and which gene or even the whole genome you want to search for. We have used it to identify proteins that might cause translation/expression problems. We, therefore, recommend using it as a first estimation tool to indicate whether your expression is working smoothly or adjustments via codon optimization or tRNA availability are necessary. We hope that the troubleshooting and prediction made for the expression will be easier.

More about the Software



Modelling

Before we begin with the expression of the genes, we wanted to know whether increased tRNA availability would lead to an increase in the translated protein. Therefore, we used an implemented gene expression model and adapted it for our purpose. The aim of this model is to fully map bacterial transcription, initiation of translation, elongation and termination. We have adapted this model, which uses ~500 ordinary differential and 7 algebraic equations, as far as possible to Vibrio natriegens. With the results obtained for the sfGFP, our model protein, we could prove that our approach of elevated tRNAs shows the potential to increase the translation of proteins with a high proportion of rare codons.

Wet lab

To provide a plasmid as a tool capable of increasing tRNA availability within Vibrio natriegens, we have thankfully received many suggestions from experts for all areas of our work: molecular biology, vector design and laboratory work.


ptRNA Design – Vector for tRNA expression in Vibrio natriegens

Thanks to the awesome discussions about our idea and the input we received during the vector design – thank you very much Dr. Altenbucher – we went for a two plasmid based approach, to be more flexible during the working and to have more selection markers. For the expression of tRNA we used a rrnA P1 promoter (from V. natriegens) and the rrnA terminator (derived from E. coli K12) and combined these with a p15A Ori in our new ptRNA. Our ptRNA carry a tetracycline resistance as selection marker.


Protein expression

To test our approach to increased tRNA availability, we planned to express sfGFP (Part:BBa_I746916) with and without different N-terminal tags with rare codons to test whether these codons affect expression. Therefore, we use fluorescence values normalized to OD600 to compare the expression levels obtained.


qPCR

In this project, we have used the powerful tool qPCR to quantify our cellular tRNA level and to prove the improved availability directly where it should be. As already mentioned, tRNA inherits a secondary structure, which leads to some problems compared to the measurement of other low-folded RNAs, e.g. mRNA, which are usually detected via qPCR. We overcome the problem by combining a modified polymerase and a specific reverse primer to amplify the desired tRNA species that are now detectable by qPCR.


Summary - Vibrio

We have provided the free and easy to use tool GeCoS to quickly get information about codons of interest in genes or whole genomes. In addition, we offer several tools to investigate tRNA availability and gain better insight into how to improve the translation of V. natriegens and take advantage of the features of this host. Our ptRNA provides the ability to increase the expression of heterologous proteins and can be used in all V. natriegens derivatives, increasing the applicability of V. natriegens. In addition, we have developed a qPCR method that allows quantitative measurement of cellular tRNA levels. This is our contribution to the establishment of V. natriegens for further applications.


Figure 1 - Two plasmid based approach to increase the expression through adjusting the cellular tRNA availability of rare codons.

Chlorella vulgaris

Introduction - Algae

Environment and sustainability are urgent topics in our generation, being important in every field. Also in biotechnology, the topic of eco-friendly processes gains more importance, characterized by the uprising of new courses of study, research questions and funding opportunities13–15, 25.


Published in 2011, the Organization for Economic co-operation and Development (OECD) pointed out opportunities and challenges for the industrial biotechnology16. Meeting market demand whilst increasing sustainability is from critical importance for the industry.


Biotechnological processes often are not only eco-unfriendly but also have often bad reputations in publicity because of the use of ethically questionable bacterial growth media. Brain heart infusion Broth, being a commonly used growth medium for Vibrio natriegens and other bacteria, is made up by infusions from calf brain and beef heart that are sources of nitrogen, carbon, growth factors, vitamins and amino acids.


Therefore, not only new environmentally friendly but also ethically reasonable bacterial growth media components must be found and used in the processes. To provide an alternative, we have therefore used the carbon fixation of a phototrophic algae as bacterial growth medium component to only not reduce the ecological footprint of the whole process but also make the process ethically acceptable. Algae are commonly used as a dietary supplement17 and can be used as a source of biofuels18.


By producing proteins, vitamins and other nutrients and using carbon dioxide and light in a process called photosynthesis19 , microalgae deliver important components for bacterial growth.


The easy cultivation of microalgae can be carried out by mainly carbon dioxide, minerals and light19,  while they produce oxygen by fixing carbon dioxide20. Some species of the microalgae Chlorella can also grow under heterotrophic conditions, using organic carbon sources like sugar21. Heterotrophic cultivation reveals the possibility of higher productivity of the microalgae22.


The algae C. vulgaris is a microalgae appearing green because of the high content of chlorophyll. Microalgae make a suitable bacterial growth media component because of its high amount of starch26, its easy cultivation and relatively fast biomass production.


Therefore, the aim was to first establish a suitable cultivation for the microalgae Chlorella vulgaris in our lab in a self-built bioreactor. The disruption of the cell wall and the measuring of the contents via HPLC analysis of the microalgae was the next step, whereas the thick cell wall of the microalgae was one of the many challenges to overcome in order to extract compounds like proteins or sugar23. After successful disruption, media based on the microalgae was produced, tested and optimized for the fast-growing organism Vibrio natriegens.


The challenges of our project lied in the establishment of algae cultivation, microalgae disruption and production of suitable bacterial media based on the extract.

Cultivation

The first step was selecting the most suitable microalgae organism for our experiments and cultivating it by reaching a fast biomass production. Talking to many experts in the field of microalgae cultivation, we choose the organisms Chlorella vulgaris and Chlorella sorokiniana because of its easy cultivation, its contents and the great knowledge that already existed about these microalgae. Comparing the productivity and the content of carbon sources such as sugars or fatty acids, we decided to focus more on the model organism C. vulgaris because of its high content of starch and its fast growth rate.


The cultivation of the microalgae started in small scale with the aim to build our own photobioreactor for large-scale biomass production. The microalgae were first cultivated in 200 mL flasks at room temperature and 200 rpm in suitable media containing salt and various trace elements. With ongoing cultivation and increasing knowledge about algae growth and biomass production we started to scale up our algae cultivation in self-made bioreactors containing 2 liters of algae suspension. We hereby established ongoing illumination by red and blue light, magnet stirring as well as gassing with room air. After another scale-up leading to a 5 and then 10 liters bottle, we decided to build an airlift photobioreactor for faster cultivation and therefore, faster biomass production. The 37 L reactor was designed and built by our team after discussion with many experts. With having six light tubes build into the reactor the airlift design is very unique and matchless compared to any other airlift photobioreactor. Due to the airlift reactor, the effective illumination surface was increased significantly, increasing biomass production and algae growth. This enabled us to follow through with our planned experiments.


Modeling

To build an airlift photobioreactor fitting our approaches of fast biomass generation of the microalgae C.vulgaris, we conducted a CFD simulation (Computational Fluid Dynamics). This was necessary in order to determine whether the integrated light tubes interfere with the airlift circulation and what velocity we can expect. Therefore, based on the preliminary specs of the planned airlift we were able to reconstruct an in-silico model. This model was then meshed in more than 737 043 tetrahedral cells with the flow direction was calculated for each cell using the navier-stokes equations for more than two weeks. The final results of our modelling proved our concept, that integration of light tubes into the reactor does not interfere with the airlift circulation and did not show any variations in the liquid flow compared to a normal airlift reactor. Thanks to the model, we hereby were able to set-up the airlift photobioreactor enabling us to increase our algae biomass production.


Cell disruption

After cultivation, the obtained biomass had to be disrupted, in order to extract the biomolecules of interest (carbohydrates and amino acids). Therefore, to determine the best and most suitable method for cell disruption, we tried several different approaches: We hereby distinguished between mechanical (High-pressure-homogenization, bead-mill and mortar) chemical (pH1, 3, 6 and 12) as well as temperature disruption (60°C, 100°C, autoclavation and microwave treatment). Furthermore, with reports of breakage and de-glycosylation of the thick cell wall of microalgae after acid treatment at a high temperature, we tested this method in combination to the mechanical disruption method of choice (bead-mill). As a measurement of disruption efficiency, we used several photometric detection assays: Anthrone-assay (allows the detection of monosaccharides in a liquid), rFAN-assay (allows the detection of free amino acids in a liquid) as well as Bradford-assay (allows the measurement of protein concentration). The most important component was the amount of carbohydrate, therefore we selected the disruption method of choice based on the anthrone-assay results. This method was a combination of acid hydrolysis (pH1) at 100°C for 1.5 hours with a subsequent mechanical disruption using the bead-mill. Optimization in terms of scale-up ability as well as time efficiency which was most important for the bead-mill disruption (typically in 2 mL tubes), we were able to use a bead-mill that can disrupt microalgae in a large scale (carries 50 mL falcons).



Analysis

For the precise determination of the amino acids and sugar quantities in our very own microalgae media we performed carbohydrate as well as amino acid high pressure liquid chromatography. For the analysis we solved the same concentration of PhyCoVi media in water as used for the bacterial growth experiments.
In terms of carbohydrates analyzation, we were able to detect glucose, fructose as well as sucrose. The highest carbohydrate concentration was exhibited by glucose, which in terms of our cell disruption confirmed our assumption that acid hydrolysis would lead to formation of monosaccharides of the high quantity of oligosaccharides in the outer cell wall of microalgae.
Furthermore, we were able to detect 18 out of the 20 amino acids (asparagine and glutamine were not detected). Some amino acids concentrations were at a very low level, which would explain why we needed peptone supplementation in order to compete with BHIN. However, during our media development we were able to optimize our cell disruption as well as extraction increasing the amino acid concentration compared to an earlier batch. With further optimization steps needed this result makes us optimistic for future use of our algae media as a standalone media.


Media

We developed our “PhyCoVi”-medium with the idea in mind to offer an alternative to the typically used yeast extract. We hereby disrupted the obtained microalgae, collected the hydrophilic phase (water containing the components of our interest) and freeze-dried said phase. We obtained a very distinct powder which we used to test bacterial growth against several established growth media as well as brain-heart-infusion (BHIN) the best complex growth media for V. natriegens. We were able to detect bacterial growth on our very own bacterial growth medium based on algae extract. Furthermore, in combination with peptone, the algae extract was able to keep up with the BHIN-media.


To get a better idea of the growth of other bacteria on our medium as well as proving the effect of our medium, we collaborated with more than five different iGEM teams by distributing our samples to them.
We therefore developed a SOP which in detail describes the experimental setup for testing our medium and sent the teams our anonymized samples of PhyCoVi medium with and without peptone supplementation as well as LB as a control. With our pre-created Excel-based analysis tool for direct presentation of the obtained results we offered a quick and reliable test of our media to other teams.
Thanks to the input of other teams as well as our own results we were able to optimize our media, improving bacterial growth, therefore increasing the competitiveness to other typical media.



Summary


With our approach, we offer a new eco-friendly media which is able to compete with the commonly used media such as LB and even with BHIN when adding peptone as a supplementation. Contrary to glucose from sugar cane, one of biotechnology’s 24 most commonly used substrates our media is import independent and does not need any arable area for the production. Furthermore, compared to BHIN we present completely ethical acceptable media were unnecessary animal death is prevented and CO2 emission is reduced. With the youth demonstrating for a more sustainable way of thinking and working we hereby offer a solution in our field of work. We were able to create an eco-friendly and ethical vegan media which does not need arable land (deforestation of the rainforest) or any high CO2 costing distribution throughout the globe. With our vision of the production of bioproducts out of sun (light) and air our project provided more than one step in the right direction.

References

  1. Algae-farm-power. Available at http://sustainablefootprint.org/algae-farm-power/.
  2. Tamiya, H. Mass Culture of Algae. Annu. Rev. Plant. Physiol. 8, 309–334; 10.1146/annurev.pp.08.060157.001521 (1957).
  3. Fan, L.-H. et al. Optimization of Carbon Dioxide Fixation byChlorella vulgaris Cultivated in a Membrane-Photobioreactor. Chem. Eng. Technol. 30, 1094–1099; 10.1002/ceat.200700141 (2007).
  4. Studies on chlorophyll formation in <italic>Chlorella protothecoides</italic> I. Enhancing effects of light and added δ-aminoIevulinic acid, and suppressive effect of glucose on chlorophyll formation. Plant and Cell Physiology; 10.1093/oxfordjournals.pcp.a074552 (1970).
  5. Chen, F. & Johns, M. R. Effect of C/N ratio and aeration on the fatty acid composition of heterotrophicChlorella sorokiniana. J Appl Phycol 3, 203–209; 10.1007/BF00003578 (1991).
  6. Cha, K. H. et al. Optimization of pressurized liquid extraction of carotenoids and chlorophylls from Chlorella vulgaris. Journal of agricultural and food chemistry 58, 793–797; 10.1021/jf902628j (2010).
  7. Kampen, W. H. in Fermentation and Biochemical Engineering Handbook (Elsevier2014), pp. 37–57.
  8. Hoq, M Mozammel & Al Mamun, Arafat & Asaduzzaman, Md & Khan, Md. Murad & Akand, Md & Khan, Shakila. (2013). Bioprocess development for eco-friendly microbial products and its impacts on bio-industry establishment in Bangladesh.
  9. Fernandes, Bruno & Dragone, Giuliano & Abreu, Ana & Geada, Pedro & Teixeira, José & Vicente, António. (2011). Starch determination in Chlorella vulgaris—a comparison between acid and enzymatic methods. Journal of Applied Phycology. 24. 10.1007/s10811-011-9761-5.