INTEGRATED HUMAN PRACTICES
Gold criteria
Cyano and Co-culture expert Daniel Ducat
In the beginning of our work with the three “Trinity” organisms we stumbled across several questions:
How should the growth media be composed?
How can we effectively use our cyanobacterium Synechococcus elongatus as a sugar producer?
How can we prevent the other organisms from overgrowing S. elongatus?
The synthesis of sugars in S. elongatus, especially aiming at a co-culture with heterotrophic organisms, has previously been shown by Dr. Daniel C. Ducat from Michigan State University. His Synechococcus cscB strain produces and exports sucrose in salt stress media. During our brainstorming sessions at the beginning of our iGEM journey, this strain was often named as a main example for the use of cyanobacteria in a co-culture. Finally, we decided to contact Dr. Ducat and asked him for advice and good ideas regarding co-cultures with cyanobacteria.
Luckily, he was on a symposium visit in Germany in May 2018 and offered to visit us and provide us with the cscB strain. In a private meeting, he suggested to use a sps (sucrose phosphate synthase) overexpressing Synecchococcus strain. Thus, we would be able to have sucrose production and export, without having to induce high salt stress in the culture media with NaCl. Although sucrose production would be less effective we could prevent the negative effects of salt stress regarding our heterotrophic bacteria and fungi.
In one of Dr. Ducat’s publications (A synthetic, light-driven consortium of cyanobacteria and heterotrophic bacteria enables stable polyhydroxybutyrate production, 2017), he used barium alginate beads to encapsulate cyanobacteria to enhance sucrose export. We were interested in using these beads as another option for co-culturing methods besides solid and liquid culture. Dr. Ducat provided us with a protocol for creating these beads and suggested to encapsulate all of our “trinity” organisms.
Most importantly, he suggested M2 as a co-culture medium to us, which is derived from the cyanobacterium medium BG11. He also provided us with the protocols which were then frequently used in our project .
In summary, Dr. Ducat helped us significantly in several ways that changed our project. At first, by providing us with the Synechococcus cscB strain which enabled us to conduct experiments with sucrose production and export in salt stress media.
Second, by introducing a co-culture medium to us which we ended up using for all of our co-culturing experiments.
We are glad to be given the opportunity to speak with a co-culture specialist and value his advice that changed our project significantly.
Integrated Human Practices with Dr. Spencer Scott
Our work with the quorum sensing system is based on research done by Dr. Spencer Scott and colleagues, who published the paper “A stabilized microbial ecosystem of self-limiting bacteria using synthetic quorum-regulated lysis” in the well known “Nature microbiology” journal in 2017.
Since we knew we would encounter some difficulties and had questions regarding this harsh project, the best choice for us was to reach out to Dr. Scott in order to improve our project.
At first we presented him our current progress and he showed himself very delighted that his PhD project was further studied by us. He also assured us his support.
Our idea was to use the same microfluidics system that the researchers used in the paper mentioned above. Since the paper does not precisely state which kind of system they used, we asked if their system was easily reproducible in our lab. Unfortunately, Dr. Scott informed us, that their lab was endowed with very sophisticated and costly microfluidics system and advised us against this setup. Instead he suggested us to use a simple chemostat.
Moreover, he suggested to try to apply the quorum sensing systems in different organisms, since the used parts should, according to him, be transferable to other organisms.
In addition to that, we encountered some issues when comparing the growth situation of the wild type co-culture with the mutant co-culture which included the quorum sensing harbouring microorganisms. He recommended to just check the overgrowth of the specific organisms in the two different situations.
Regarding this, another important advice he gave us was that a good overall experimental time is 24 hours.
Finally, to solve the problem of quantifying AHL - the quorum sensing molecule of interest - he proposed to us to use a qualitative approach instead of a quantitative one, hence to try titration of different AHL concentrations and to check the impact it has on our cultures.
When getting to know Dr. Scott, we found out he was a member of the iGEM team Berkeley 2011 which is why he did not only give us significant advice for our project, but also told us about his experience with iGEM, further increasing our enthusiasm for iGEM and the final Giant Jamboree.
We would like to thank Dr. Scott for this amazing skype interview and the great input he gave our team. We hope - with the gained iGEM experience and future research we will do a great job and one day will also be able to help other iGEM teams and play a role in their interesting projects.
Interview with Shraddha Shitut
Shraddha Shitut (Sh) is currently working as a postdoc at the Leiden Institute of Biology and institute of Chemistry in the Netherlands. In her PhD she worked on cross-feeding in bacteria resulting in a publication "Metabolic coupling in bacteria" in bioRxiv 114462 with which we found many similarities to our subproject "Auxotrophy". There she describes the establishment of a co-culture working with unidirectional cross-feeding with amino acids like lysine, histidine and tryptophan. We asked her for advice and wanted her to give an opinion on our approach.
TD: Hello Shraddha! We are iGEM team Duesseldorf 2018 (TD) and want to know, if you would consider giving us an interview and advice for one of our subprojects. For this year’s project we are working on establishing two- and three-way co-cultures between Escherichia coli and Saccharomyces cerevisiae and the cyanobacteria Synechococcus elongatus. One of our approaches is establishing a cross feeding of amino acids between a leucine-auxotroph E. coli and a lysine-auxotroph S. cerevisiae; they are producing the needed amino acid for each other. What is your first impression about our plan?
Sh: It is an ambitious plan. If we only consider the 2-way interaction between the modified E. coli and BY4742 you need to remember the additional auxotrophy in BY4742 and provide histidine in the medium. This may affect the dependency of the auxotrophic E. coli strain. In my experience in some situations the auxotrophy can be fulfilled even by the presence of other amino acids in the medium. Hence it would be ideal to test of all auxotrophs that are to be used in the project in the presence of the different amino acids to be used in the project. There should be a clear difference here - growth when focal amino acid is provided, no growth when any other amino acid is provided.
TD: Exactly. Our Auxotrophy system is used in a two way co-culture. We have already added histidine but didn’t consider that it could influence the dependencies. So, thank you for this tip! Why did you start researching auxotrophies and co-cultures?
Sh: I got interested in studying the interactions between bacteria (in a social or community context) during my studies for my masters degree in microbiology. Then I further read about metabolic dependencies in microbial communities and how this possibly affects the percent of bacteria we can cultivate and isolate. It was fascinating to learn how auxotrophies can lead to interactions and networks between microorganisms.
TD: Interesting! Where can this focus on co-cultures built on auxotrophies lead to in research?
Sh: Auxotrophies can be thought of as the dependency of an organism on an external source of an essential molecule which in most cases are amino acids, vitamins or nucleotides. These metabolites have to be part of the primary metabolism in a cell and hence essential for growth and proper cell function. A co-culture or interaction based on the exchange of such essential metabolites makes the interaction itself obligatory for the involved partners and hence a good model system to study. Furthermore due to the involvement of the metabolites, especially amino acids, in primary metabolism one can observe direct effects of the interaction (taking place in co-culture) on the behavior of both partners.
TD: Do you have experiences with amino acid exchange without exporters?
Sh: The cross-feeding system that I used and studied for my PhD did not inherently contain mutations in any of the amino acid exporters. We observed exchange of amino acids via physical structures connecting cells. However we cannot completely rule out the involvement of exporters as a response to amino acid starvation.
TD: In your publication it is mentioned that the exchange took place via nanotubes. Did you modify your cells for this? And if yes, how?
Sh: We used a synthetically engineered cross-feeding system wherein we introduced two mutations in wildtype
TD: We are working with a lysine auxotrophic system. Can you explain why lysine, histidine and tryptophan are good candidates to work with ? Why are they so often in focus of studies?
Sh: Lysine, histidine and tryptophan are usually selected for their isolated biosynthetic pathways which allows for easy manipulation without affecting the production of other amino acids.
TD: What were the challenges you faced and what do you think could be challenges with our approach?
Sh: The first challenge of your project is to introduce the auxotrophies and amino acid production/secretion mutations in the different partners. The second would be to ensure that the produced amino acids are sufficient to support growth (Keep in that due to the fact that the partners belong to different classes their amino acid requirement levels will likely be different). The third challenge is to successfully cultivate the 3 organisms such that each partner remains stable over a time period long enough for you to study the interaction. Here you need to take into consideration differences in growth rates, nutritional requirements, ambient pH and so forth for the 3 organisms.
TD: What methods did you use to quantify your amino acid production?
Sh: I used both an indirect biosensor based approach and a chemical analysis technique (LC-MS) for quantifying amino acid production.
TD: What method would you suggest if we want to extract amino acids from the cells?
Sh: Internal amino acid extraction requires two main steps - breaking open the cell and purifying the amino acids within. The cell wall and membrane can be disrupted either by physical techniques (sonication, heat, beads) or by chemical techniques (lysozyme). The choice of techniques is dependent on factors like equipment availability in the lab, number of samples to be purified at a given time, characteristics of the cell (i.e. cell wall thickness) and so on. The second step of purifying the amino acids from the cell extract also differs based on which amino acids are of interest (free amino acids, protein bound amino acids).
The method I used in my study was based on a physical disruption of E. coli cells using heat and sonication followed by solvent based extraction of internal free amino acids. This method is useful for extraction of internal amino acids from numerous and multiple samples.
TD: Did you also measure the composition of your cultivation media to know how much of the produced amino acids are exported? If you did, can you suggest a method to use here as well?
Sh: I was using minimal media for all my experiments and during the chemical analysis of my samples for amino acid quantification I always used asterile control media. This was especially important because minimal media contains ammonium chloride as a nitrogen source and the NH-group here interferes with LC-MS measurements. More specifically because we were derivatizing (using FMOC to basically label the amino groups to make it detectable in the machine) the samples prior to amino acid quantification and we had to add excess reagent to first nullify the NH-groups already present in blank minimal media.
TD: We are working on a co-culture with different organism classes. Did you use organisms other than E. coli and what are your experiences?
Sh: We used Acinetobacter baylyi in the cross-feeder co-cultures with E. coli. A. baylyi is a gram negative soil dwelling bacteria hence not very different from E. coli in terms of amino acid biosynthetic pathways and lab cultivation. It was however tricky to introduce the auxotrophic and over-production mutations in A. baylyi as compared to E. coli. This (genetic manipulation) I think is the biggest hurdle in organisms that are less established than E. Coli, followed by finding the right media that supports the growth of all partners.
TD: What do you think are the advantages of co-cultures and co-cultivation in general?
Sh: I think co-culture and co-cultivation approaches can give us a lot more information about bacterial behavior than isolation based techniques. By culturing bacteria with other organisms we introduce a factor in its environment that can result in a number of reactions or interactions, both positive and negative. Hence such techniques can lead to novel compounds being produced by the involved bacteria.
TD: Thank you very much, for your precious advice and time. You helped us a lot!