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Our work with Azotobacter vinelandii Why Azotobacter vinelandii We chose Azotobacter vinelandii for its ability to produce the extracellular biopolymer alginate. In particular vegetative DSM 576 (or NCBI 9068), which provides a high alginate production.3 , 6 Pseudomonas mendocina in mind, but due to concerns regarding the properties of its alginate (further explained in the “alginate ” section), we decided against it. The biopolymer alginate is secreted by A. vinelandii to protect the enzyme Nitrogenase against oxygen during its vegetative growth, but also to protect it from heavy metals2
How to start We obtained A. vinelandii (DSM 576) from the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen / German collection of microorganisms and cell cultures). The culture was delivered as dried cell pellet. We opened, rehydrated and inoculated the bacteria according to the delivered protocol . We cultivated A. vinelandii in the “Azotobacter medium" (Medium 3 ) and the Burk’s medium.
Cultivation The most used medium by us was the Burk’s medium. A big advantage of this medium is that you have two solutions: one simple buffer and a high concentrated solution of nutrients. This can be useful as with 1l of stored nutrient solution one can make 10l medium and the buffer can be done quickly. We experienced no contamination in those solutions.
As only nitrogen fixing organisms can grow in this medium, we can conclude the successful cultivation of A. vinelandii
The cultures were incubated at 30°C according to the preferences of this strain.2
Modelling Note: This text only gives a short insight on the methods we used to build our metabolic model of A. vinelandii . Further information can be found under “Modelling ”.
We cultivated A. vinelandii in 3 different flasks. The flasks were grown for ten days. Each day, 21 samples (3x for dry cell mass, 3x for sucrose, 1x for alginate-measurement for each culture) in total were taken to measure different contents in the culture. We wanted to obtain data for the dry cell mass, the sucrose content in the medium and the alginate production.
Dry cell mass measurement The dry cell mass was determined by weighing the dried sample. We measured the empty tubes before filling them, to get more precise weights and avoid scraping the dried pellets out of each tube. We also measured the optical density (OD) at the wavelength 620 nm to detect bacterial growth. This way, we were able to establish a correlation between the OD and the dry weight, which was important for our model.
Saccharose measurement To measure the sucrose content (carbon source for A. vinelandii ), we used a Sucrose Assay Kit (ab83387, abcam). With this kit, the remaining sucrose is converted into glucose and a “reaction mix” makes this concentration measureable by OD at a wavelength of 570 nm. A higher sucrose concentration results in a higher OD. Then the OD can be compared with an established standard curve of known Sucrose concentrations. With these measurements, we could provide important information for our model, for example the amount of carbon source needed for a specific alginate concentration.
Alginate measurement For measuring the alginate content, we measured all carbohydrates in the cell pellets to quantify the extracellular alginate concentration. By mixing each sample with the chemical anthrone and comparing the samples to a standard that has been created, we were able to estimate the alginate concentration. For further explanation, we followed a protocol 9
Electroporation The stored A. vinelandii were made electrocompetent before they were stored at -70°C. The matching Eppendorf Multiporator protocol was applied, the cells were washed four times with glycerol. Each wash step was followed by centrifuging the cells at 4,500 rpm for 10 min. After this, aliquots were frozen in liquid nitrogen and stored at -70°C.
Liquid cultures, from a dense Burk’s medium, were mixed in a ratio 1:4 or 1:5 with 50% autoclaved Glycerol up to 1 mL and stored at -70°C in cryovials.
An alternative way to store
A. vinelandii can be found in the “research ” section.
High GC-content While designing our construct for cellulose synthesis, we faced the challenge of getting our hands on the actual genes from Komagataeibacter xylinus because many service providers were not able to synthesize them due to their high GC content. To overcome this problem, we tried to change the codons manually to lower the GC content, but we could only decrease it by ~5%. We were aware that the codon optimization for A. vinelandii could be destroyed during this process and the actual expression rate could differ from the natural expression rate.
We found out that the iGEM distribution kit contains some genes we wanted to use for our cellulose synthase construct. So we adjusted our cloning plan and designed primers with overhangs for a Gibson assembly and wanted to link them together later. For the missing genes we found a provider willing to synthesize these smaller parts with high GC-content
Our research For our project, we had to do a lot of research on how to handle A. vinelandii in the lab. Here we present the most important information we considered during our work. In order to improve the work with Azotobacter for future iGEM teams, we hope to give an overview on how to handle A. vinelandii in the lab. Because it was a big part of our project, we will also present the mechanism of the alginate biosynthesis in a section itself.
Alginate Alginate is a bacterial biopolymer which consists of two subunits: D-mannuronic acid and L-guluronic acid. They are linked to each other by glycosidic bonds. Alginate can vary in length and proportion of its subunits. This is why the molecular weight also varies when alginate is produced by different organisms. The subunit L-guluronic acid forms a more stable alginate polymer with a higher viscosity in combination with divalent ions like Calcium ions (Ca2+ ).2 A. vinelandii produces more L-guluronic acid subunits compared to our other option Pseudomonas 2 A. vinelandii for our project, because we wanted to produce a stronger polymer for our bioink.
A precursor of alginate is GDP-mannuronic acid, which is synthesized from Fructose-6P, and polymerized at the inner membrane by the Glycosyltransferase (alg8), the key enzyme in our project. By overexpression of alg8 , we aim to increase the alginate secretion, because it has been proven that this can have a positive effect on alginate synthesis.2
Figure 1, alginate synthesis2 Strain DSM 576 The DSMZ recommends two different media for our strain: Medium 3 and Medium 441 . In both media, A. vinelandii should be cultivated at 30°C 3 Azotobacter medium” is more simple to prepare than medium 441, the diazotrophic medium (RBA). This is why we chose medium 3 to culture A. vinelandii first. Both media lack a nitrogen source, which indicates that a successful cultivation of microorganisms in these can be considered as proof for nitrogen fixing bacteria.
The most common medium used when working with A. vinelandii was the Burk’s Medium: It is divided into two individual stock solutions that have to be mixed in a 1:10 (nutrient solution - phosphate buffer) ratio.5
10x Nutrient solution Component Amount Sucrose 200 g MgSO4 7H2 O 2.0 g CaCl2 2H2 O 0.9 g 10 mM Na2 MoO4 H2O 1 mL FeSO4 7H2 O 50 mg dH2 O up to 1 L
Phosphate buffer Component Amount KH2 PO4 0.2 g K2 HPO4 0.8 g dH2 O up to 900 ml
Since it was our goal to produce alginate for out bioink, we searched for publications that also wanted to produce biopolymers in A. vinelandii and found: “Alginate production and alg8 gene expression by A. vinelandii in continuous cultures” by Díaz-Barrera et al (2012)4 alg8 -overexpressed) alginate production on a larger scale. The medium differs from other A. vinelandii -media because it contains nitrogen4
Medium for A. vinelandii Compenent Amount Sucrose 10, 15 or 20 g (NH4 )2 SO4 0.8 g K2 HPO4 0.66 g KH2 PO4 0.16 g CaSO4 0.05 g NaCl 0.2 g MgSO4 7H2 O 0.2 g Na2 MoO4 2H2 O 0.0029 g FeSO4 0.027 g
Exact introduction on how to prepare this medium can be found in the original paper , cited and listed in “sources” - Nr 4. A. vinelandii will produce more alginate when not grown under nitrogen starvation in this setup.4
In order to achieve optimal alginate production, the publication “Optimal conditions for alginate production by Azotobacter vinelandii ” already published the best conditions necessary for A. vinelandii . They used the A. vinelandii strain DSM 576, like we did in our project, so this medium is mentioned here to complete the information we had on this strain. The main difference to the other media listed here is that they used yeast extract.6
Media for storage, competency or transformation are well described in the paper “Molecular Biology and Genetic Engineering in Nitrogen Fixation”5 protocols ”, where we describe some methods used on A. vinelandii that may be important when working with it.
Antibiotics Since it was our plan to genetically engineer A. vinelandii , we needed to find standard antibiotics and their recommended concentrations for a direct selection. Since A. vinelandii is not the most established organism in iGEM so far, we asked Leo Chi U Seak, who already worked with A. vinelandii and he gave us a lot of good information we will display here.
Antibiotic Concentration Reference Ampicillin 50–100 μg/mL Page WJ, Grant GA (1987) and Bertsova YV, et al (2001) Kanamycin 0.5–3 μg/mL Peralta-Gil M, Segura D, Guzman J et al (2002) Tetracycline 10 μg/mL Kelly MJ, Poole RK, Yates MG et al (1990)
This table is shortened to the antibiotics we had in storage/easy access to and the way the references were displayed changed.
An iGEM report from 2016 (PLOS Blog) also claims that A. vinelandii already stops growing under ampicillin concentrations of 10 µg/ml 1
Protocols To genetically engineer A. vinelandii , they can be grown under iron starvation in the “Competent medium”. It is exactly the same as the “Burk’s Medium”, but does not contain FeSO4 and Na2 MoO4 . First, A. vinelandii is grown on a “Competent medium” agar plate for at least two days, then inoculated in a liquid culture and incubated for up to 20 h. The DNA can be simply added into this culture and A. vinelandii will take it up. Including the selection, where the transformed cells grow on an agar plate with a matching antibiotic resistance, which also takes up to 20 h, this process will take up to five days if you do not have competent cells in stock.
In order to integrate the DNA into the genome of A. vinelandii , it is recommended to use at least 150-500 bp matching/flanking sequences.5
One of our Advisors, who also worked with A. vinelandii, provided us with an alternative way for transformation: Electrocompetent cells have to be incubated for one minute with 100 ng DNA and put into an electroporation cuvette. The electroporator settings are the same as for E. coli (1.8 kV pulsating, BioRad MicroPulser). The cells can be put in 500 µL Burk’s medium and incubated for 1 hour at 30°C. After incubation, the electroporated cells can be selected on an agar plate with the matching selection marker.
Cell growth measurement The cell growth can be tested by measuring the OD. Before it is possible to use this method, a correlation between OD and the dry cell weight has to be created.4 620 for our modelling approach, while here the OD450 was used. The correlation between cell growth and OD620 has already been established by us and can be seen in the section “Modelling ” of this project.
Storage As an alternative way to storing A. vinelandii in the -70°C freezer (“Our work with A. vinelandii ” - “Storage”), we had a protocol from Markus W. Ribbe on hand. To store A. vinelandii , a picked culture from an agar plate can be put in a “Storage buffer'', which is a simple phosphate buffer, like the one used for the Burk’s medium, with 1% DMSO (Dimethylsulfoxid). 1 mL of this buffer for each colony can be stored at -80°C 5
Potential We decided for us that A. vinelandii was the best microorganism to produce biopolymers for our project, but it also has other properties that may be useful for other iGEM teams. As a biologist, it is always important to keep in mind that E. coli can not represent the microbiological diversity and that there are certain properties and abilities other bacteria can perform better.
WWe hope that our contributions/information can give a basic idea of working with A. vinelandii and spark creativity for future iGEM projects.
A. vinelandii is a nitrogen fixing bacteria. The most important enzyme, the nitrogenase, already has been a focus of studies on this complex process.5 The genetics of A. vinelandii are strange. It has been proven that A. vinelandii can have a high number of chromosomes, which depends on the growing conditions.7 Since A. vinelandii is a soil bacterium, which can produce hydrogen and fix nitrogen, it has potential in improving agriculture. There have already been investigations on how big the advantages can be.8 Sources: https://collectionsblog.plos.org/igem-report-004/ [last opened: 07.10.2022]Urtuvia, V., Maturana, N., Acevedo, F. et al. Bacterial alginate production: an overview of its biosynthesis and potential industrial production. World J Microbiol Biotechnol 33, 198 (2017). https://doi.org/10.1007/s11274-017-2363-x https://www.dsmz.de/collection/catalogue/details/culture/DSM-576 [last opened: 07.10.2022]Alvaro Díaz-Barrera, Erik Soto, Claudia Altamirano, Alginate production and alg8 gene expression by Azotobacter vinelandii in continuous cultures, Journal of Industrial Microbiology and Biotechnology, Volume 39, Issue 4, 1 April 2012, Pages 613–621, https://doi.org/10.1007/s10295-011-1055-z Ribbe, Markus W. (2011). [Methods in Molecular Biology] Nitrogen Fixation Volume 766 || Molecular Biology and Genetic Engineering in Nitrogen Fixation. , 10.1007/978-1-61779-194-9(Chapter 6), 81–92. https://doi.org/10.1007/978-1-61779-194-9_6 Francesca Clementi, Paolo Fantozzi, Francesca Mancini, Mauro Moresi, Optimal conditions for alginate production by Azotobacter vinelandii , Enzyme and Microbial Technology, Volume 17, Issue 11, 1995, Pages 983-988, ISSN 0141-0229, https://doi.org/10.1016/0141-0229(95)00007-0 Nagpal P, Jafri S, Reddy MA, Das HK. Multiple chromosomes of Azotobacter vinelandii . J Bacteriol. 1989 Jun;171(6):3133-8. https://doi.org/10.1128/jb.171.6.3133-3138.1989 . Erratum in: J Bacteriol 1989 Nov;171(11):6395. PMID: 2785985; PMCID: PMC210026. Barney, B.M. Aerobic nitrogen-fixing bacteria for hydrogen and ammonium production: current state and perspectives. Appl Microbiol Biotechnol 104, 1383–1399 (2020). https://doi.org/10.1007/s00253-019-10210-9 Ashley E. Beck, Kristopher A. Hunt, Ross P. Carlson, Measuring Cellular Biomass Composition for Computational Biology Applications, 24 April 2018, https://doi.org/10.3390/pr6050038 Joshua M. Pearce, Bas Wijnen (2016). "Open-source syringe pump". Appropedia. Retrieved October 13, 2022 https://www.appropedia.org/Open-source_syringe_pump#How_to_Build_an_Open-source_Syringe_Pump [last opened: 13.10.2022]