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

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.

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.

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.

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 shouldn't 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 as there is plenty of Arginine 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 pickable 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 pickable. 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 8 shows the results of the WT transformation under these conditions.

A KO of an essential gene from Synechocystis took more time than expected. However, with more time this is 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. In addition we would be able to fully test our consortia and verify that E. coli alone is capable of providing enough Arginine for an ΔargH mutant Synechocystis.

Parts submitted to the registry include the construct used for step two of the transformation protocol. This removes the mazF/aphII cassette from the genomic DNA such that there is a markerless knockout of argH in Synechocystis