In our consortia we strive for sustainable and stable processes. Synechocystis does not need anything but water and sunlight to survive however this implies it cannot benefit from E. coli's presence in our consortia. Therefore, in this module we aim to engineer an auxotrophic Synechocystis that is dependent on nutrient produced by E. coli.
We want to engineer a productive relationship. As such, a relationship in which the survival of one organism is directly tied to the survival of the other can have a beneficial impact. An auxotrophic Synechostishas many benefits in our consortia, which are explained in this section.
How does one make a KO of an essential amino acid or vitamin in Synechocystis? In this section we explain the proposed KO protocol adopted in our project
We were able to make KO constructs that have been shown to be able to successfully create an auxotrophic Synechocystis.
One can construct a synthetic consortium between two species with a variety of strategies. Synechocystis could simply be engineered to export a carbon source (such as glucose) for E. coli in a glucose-absent medium. Although such a commensal relationship would definitely under certain conditions be considered stable, E. coli alone is the benefactor - there is no drive for Synechocystis to produce this carbon source. Increased stability is possible by creating a reciprocal relationship between the two species. Therefore, how can Cyanobacteria benefit or depend on E. coli’s presence? Cyanobacteria manufacturers its own food and has grown for millions of years requiring only sunlight and water, in addition to inorganic elements. In fact, E. coli’s presence actually harms Cyanobacteria by possibly limiting access to precious sunlight. Herein lies this module’s focus: Genetic engineering of an E. coli dependent Synechocystis for the formation of stable synthetic consortia.
A basic requirement of consortia is the ‘communication’ between the species. Here we will attempt to construct a mutualistic relationship in which survival of one species is obligatory and beneficial for the survival of the other. The mode of communication will be the mutual production and exchange of essential metabolites. Since Synechocystis is an autotroph, one way to realize such a co-dependency is through the engineering of an auxotrophic bacterial strain. E. coli, could then in exchange produce the nutrient Synechocystis has been engineered to require. Ultimately, Synechocystis will produce a carbon source necessary for survival of E. coli, while E. coli produces a nutrient necessary for survival of Synechocystis - an engineered interdependence pathway.
This module will aim to engineer an auxotrophic Synechocystis. It also aims to test this auxotroph in the presence of an E. coli engineered to produce the nutrient that Synechocystis has been engineered to be deficient in. One might ponder what is the added benefit of this feedback system in our consortia. Indeed, as stated before, stability can be achieved with just Synechocystis feeding E. coli. However, with this feedback loop, we gain numerous benefits over the aforementioned system.
- Considering containment of GMO, an E. coli dependent Synechocystis would not be able to survive outside the lab, thus the risk of environmental biocontamination would be greatly diminished.
- An essential part of our project involves the creation of an emulsion based protocol to test potential consortia. If Synechocystis did not require E. coli, testing out the most effective and stable consortia in this manner would result in simply selecting against the presence of E. coli, as Synechocystis would do whatever it takes to increase it’s own numbers. By having a feedback loop, we are therefore able to select consortia in which the members work well together.
- Assuming that E. coli could produce the required compound (e.g. an amino acid such as arginine or a vitamin) faster than Synechocystis can itself - by knocking out the gene that encodes the enzyme to produce this compound, Synechocystis does not need to spend energy in these pathways.
There are several criteria that need to be upheld when making the choice of what sort of auxotroph should be made. In our modeling efforts, a novel algorithm was developed to searched the metabolic map of Synechocystis and output candidate nutrients that fulfilled the following criteria:
- Simple. We want to choose a nutrient that can be relatively simple to knock out. Thus in our search, we focused on nutrients that only required the knock-out of one gene to produce the desired phenotype.
- Loss of the gene encoding the reaction to produce the nutrient would result in no photoautotrophic growth. If the nutrient is not in the medium, Synechocystis should not grow.
- Reintroduction of the nutrient to the medium would result in growth of Synechocystis.
- The nutrient should be able to be produced and secreted by E. coli
A novel constraint based modeling algorithm, the auxotroph finder, was developed within this iGEM endeavour (link here) with the afore-mentioned criteria in mind. It was able to find a variety of different suitable nutrients for which would be suitable candidates. Of these, two amino acids were chosen to be knocked out: arginine and proline.
arginine was considered to be a particularly suitable target as we found a strain of E. coli that was able to synthesize it. In addition it has been shown that Synechocystis cells can grow faster in the presence of arginine. The enzyme responsible for arginine production in Synechocystis is L-arginosuccinate lyase (ASL) enzyme. This is predicted to be encoded by the slr1133: argH gene. ASL drives the reaction from argininosuccinate into arginine and fumarate in the Urea Cycle. Figure 1 displays the pathway leading to arginine synthesis.
In the case of proline, a strain of Salmonella and E. coli capable of producing proline was procured. The enzyme responsible for proline production in Synechocystis is pyrroline-5-carboxylate reductase (P5C reductase). This is predicted to be encoded by the slr0661: proC gene. Figure 2 displays the pathway leading to proline synthesis.
Figure 1. - Production of arginine in Synechocystis is associated with the Urea cycle. ASL, circled in red, is the gene of the enzyme whose loss results in a arginine auxotrophic Synechocystis
Figure 2. - Production of proline in. P5C reductase , circled in red, is the gene of the enzyme whose loss results in a proline auxotrophic Synechocystis
To conduct an auxotrophic Synechocystis, we followed the markerless knock-out protocol developed by Albers et al. This method involves a two step transformation of Synechocystis. During the first transformation, a cassette including a gene encoding antibiotic resistance is inserted into the genomic DNA, replacing the gene of interest. In the second transformation, counter selection under nickel yields colonies that have lost the inserted cassette. Overall resulting in a markerless transformation of Synechocystis to be deficient of the gene of interest. Figure 3 displays the overall mechanism of this protocol. Two plasmids were used. The plasmid shown in 4a contains the mazF/aphII casette. aphII is a gene encoding for Kanamycin resistance. nrsR--nrsS detects nickel. PnrsB is a nickel induced promoter. mazF is a protein synthesis inhibitor. In the presence of nickel, nrsR-nrsS detects the nickel, induces the PnrsB promoter which expresses mazF - inhibiting cell growth. In the presence of Kanamycin, cells without this cassette will die. Plasmid shown in 4b) is Ampicilin resistant plasmid and contains the up and downstream homologous regions to the gene of interest.
Synechocystis was cultured on BG11 (with specific variations indicated below). For all transformations used in this module, BG11 was supplemented with 50 ug/ml of Kanamycin.
Synechocystis has multiple copies of its genome contained within several identical chromosomes. Therefore after Synechocystis transformation, it is necessary to verify that the insert/deletion is present in all copies. If not, the WT phenotype could easily take over. If the insert/deletion is not present in all copies, a process called 'segregation' is used to add further selection pressure to Synechocystis. During segregation, Synechocystis is grown in the presence of kanamycin over a long period of time. Frequent colony PCR checks are used to check the genomic status of the transformation.
Figure 3: Overall Mechanism of counter selection protocol. A) Plasmid containing up/downstream homologous region and mazF/aphII cassette is transformed into Synechocystis. Selection on Kanamycin leads to the gene of interest to be replaced by the mazF/aphII cassette in the genome. B) Synechocystis is then transformed again with the plasmid containing up/downstream homologous regions to the gene of interest. Counter selection on nickel leads to the loss of the mazF/aphII cassette.
To create a strain of Synechocystis auxotrophic in arginine, we chose to knock-out the argH gene. We were able to make both knock-out constructs depicted in Figure 3. Verification of the constructs via Colony PCR of the transformed E. coli colonies are shown in Figure 4.
After the constructs were made, transformation of Synechocystis was attempted using BG11 supplemented with 1 mM arginine and 50 ug/ml Kanamycin. Two strains of Synechocystis were transformed: WT strain and the Δacs acetate producing strain. [See Carbon producing module] After 2.5 weeks, PCR of resulting colonies suggested no presence of the mazF/aphII cassette (Figure 5 for either strain in the genomic DNA.
Very limited progress was made regarding the construction of a proline auxotroph Synechoystis. Although we were able to amplify the downstream homologous region of proC gene using a DNA polymerase with proofreading (Herculase), the upstream homologous region proved more difficult to amplify. Various troubleshooting attempts were implemented but ultimately only the Taq polymerase was capable of amplifying the region as shown. Fusion was then conducted with this Taq amplified upstream fragment and the Herculase amplified downstream fragment, but this failed. One possible reason for the inability to use Herculase polymerase to amplify this region might be due to the choice of primers. Due to the issues with amplifying this fragment, proline was put on hold in favor of focusing cloning activities on the argH knockout.
Because the expected bands were not present in the colony PCR verification and the gene was still very much in the genome, additional checks were conducted. Synechocystis should not have been able to survive with the kanamycin added to the medium, so specific primers especially for the mazF/aphII cassette were used to check whether it was inserted. This check proved that mazF/aphII was transformed into the genome but still did not knock out the required gene. A check for single crossover was conducted that yielded negative. The same transformation was repeated again with similar results.
A different transformation was employed to improve the probability of success of Synechocystis to lose the argH gene. Cells were grown on BG11 medium with or without nitrate added, in addition to supplementation with 5 mM of arginine. The rationale behind growing with and without nitrate is so that for Synechocystis cells grown without nitrate - arginine would be the only nitrogen source, therefore forcing the cells in a position that it is more favourable for them to lose the argH gene since they had to rely already on Arginine made avilable in the medium. In addition to these changes, the protocol was altered such that during all stages of transformation arginine 5mM would be present in the medium, including when inoculating cells with the KO construct. These changes were all implemented and used to transform both WT and the Δacs acetate producing strains. Only the WT strain grown on nitrate resulted in single isolatable colonies. Both Δacs strains grown with and without nitrate in the BG11 as well as the WT strain grown without nitrate resulted in overgrowth of the culture such that no colonies were isolatable. This was a strange result that given the time - would have been more strongly investigated. Nevertheless, conducting the transformation in this way yielded much more promising colonies in terms of insertion of the mazF/aphII cassette. Figure 6 shows the results of the WT transformation under these conditions.
Figure 5: Gel of colony PCR after transformation of Synechocystis. Upstream forward and downstream reverse homologous region argH primers were used. WT untransformed genome is shown on first well as a control. Size of the argH gene is 1.5 kb. 3.5 kb band seen in all wells signifies the presence of the gene (1.5 kb + 2kb homologous region). 5.7 kb band indicating the presence of mazF/aphII cassette is not seen in any of the colonies
Figure 6: Gel of colony PCR after altered protocol transformation of Synechocystis. Upstream forward and downstream reverse homologous region argH primers were used. WT untransformed genome is shown on first well as a control. Size of the argH gene is 1.5 kb. 3.5 kb band seen in all wells signifies the presence of the gene (1.5 kb + 2kb homologous region). 5.7 kb band indicating the presence of mazF/aphII cassette is seen in many of the colonies
A KO of an essential gene from Synechocystis was achieved but as expected, the timeframe of this project did not allow for the isolation of a fully segregated markerless construct. However, given more time, we provide strong evidence that this should be definitely possible. The colonies that looked most promising from our gel would undergo segregation such that for all copies of genome, argH gene would be removed. After this is confirmed, the second step of transformation via nickel selection would be performed. Removal of mazF/aphII cassette would be verified via colony PCR. Segregation under intense nickel pressure would also be performed if necessary. Once a suitable markerless knockout strain has been verified, testing would be conducted to confirm that this strain would be unable to grow on media not containing arginine. Subsequently, we would be able to test whether in our consortia that E. coli alone is capable of providing enough arginine for an ΔargH mutant Synechocystis to grow.
Malcolm Watford, Glutamine Metabolism and Function in Relation to Proline Synthesis and the Safety of Glutamine and Proline Supplementation, American Society for Nutrition, 2008
Cheah, Y. E., Albers, S. C. and Peebles, C. A. M. (2013), A novel counter-selection method for markerless genetic modification in Synechocystis sp. PCC 6803. Biotechnol Progress, 29: 23–30. doi: 10.1002/btpr.1661