Difference between revisions of "Team:Amsterdam//Project/Synthetic biology/Dependecies"

One can construct a synthetic consortium between two species with a variety of methods. 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 it’s own food and has grown for centuries requiring only sunlight and water, in addition to inorganic elements. In fact, E. coli’s presence actually harms Cyanobacteria by 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 requires ‘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. This is visualized in Figure 1. 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.

1. We consider potential outbreak risks. An E.coli dependent Synechocystis would not be able to survive outside the lab, thus the risk of environmental contamination would diminish.
2. 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 growth rate. By having a feedback loop, we are therefore able to select consortia in which the members work well together.
3. Assuming that E. coli could produce Arginine faster than Synechocystis can itself. By knocking out the gene to encode for the enzyme to produce arginine, Synechocystis does not need to waste energy in making these enzymes.

There are several criteria that to be upheld when making the choice of what sort of auxotroph should be made. In our modeling attempts, an algorithm was developed to searched the metabolic map of Synechocystis and output candidate nutrients that fulfilled the following criteria.

1. 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.
2. Loss of the gene encoding the reaction to produce the nutrient would result in non-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.
3. The nutrient should be able to be produced and secreted by E. coli.

With the aforementioned criteria in mind, the auxotroph finder that was developed by one of our team members 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 a 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 encoded by the slr1133: ArgH gene. ASL drives the reaction from argininosuccinate into arginine and fumarate in the Urea Cycle. Figure 2 displays the mechanism behind Arginine production.

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. This is encoded by the slr0661: ProC gene. Figure three displays the mechanism behind Proline production in 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 who have lost the inserted cassette. Overall resulting in a markerless transformation of Synechocystis to be deficient of the gene of interest. Figure 4 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.

Figure 5: Overall Mechanism of counter selection protocol. B) 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. C) 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.

Synechocystis grows on BG11. For all transformations used in this module, BG11 was supplemented with 50 ug/ml of Kanamycin.

Synechocystis has 12 copies of its genome contained within one chromosome. Therefore after Synechocystis transformation, it is necessary to verify that the insert/deletion is present in all genomic copies. If not, the WT phenotype could easily take over. If the insert/deletion is not present in all genomic 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.

Although we were able to amplify the downstream homologous region of ProC gene using a DNA polymerase with proofreading (Hercules), 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 Hercules amplified downstream fragment, but this failed. One possible reason for the inability to use Hercules 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.

To create a strain of Synechocystis auxotrophic in Arginine, we attempted to knock-out the argH gene. We were able to make both knock-out constructs depicted in Figure 4. Verification of the constructs via Colony PCR of the transformed E. coli colonies are shown in Figure 9.

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 10) for either strain in the genomic DNA.