Matching Items (2)
Description
Traditional methods of genetic engineering are often limited to relatively few rounds of gene additions, deletions, or alterations due to a lack of additional available antibiotic resistance markers. Counter-selection marker methods can be used to remove and reuse marker genes as desired, resulting in markerless engineered strains and allowing for theoretically unlimited rounds of genetic modifications. The development of suitable counter-selection markers is vital for the development of model organisms such as cyanobacteria as biotechnological platforms.
In the hopes of providing other researchers with a new tool for markerless genetic engineering of cyanobacteria, the toxin MazF from E. coli was developed as a counter-selection marker in the most widely used cyanobacterium, Synechocystis sp. PCC 6803. The mazF gene from E. coli was cloned and inserted into a plasmid vector for downstream transformation of Synechocystis. The plasmid construct also contained two homologous flanking regions for integration of the insert into the Synechocystis genome, a nickel-inducible response regulator and promoter to control MazF expression, and a kanamycin resistance gene to serve as the antibiotic marker. In order to ensure the mazF plasmids could be cloned in a MazF-sensitive E. coli host even with slight promoter leakage, MazF expression was toned down by decreasing the efficiency of translation initiation by inserting base pairs between the ribosome binding site and the start codon of the mazF gene. Following successful cloning by E. coli, the mazF plasmids were then used to transform Synechocystis to create mazF mutant strains. Genomic analysis confirmed the successful transformation and segregation of mazF mutant strains containing the desired marker cassette. Phenotypic analysis revealed both growth arrest and production of mazF transcripts in mazF mutant strains following the addition of nickel to the cell cultures, indicating successful nickel-induced MazF expression as desired.
In the hopes of providing other researchers with a new tool for markerless genetic engineering of cyanobacteria, the toxin MazF from E. coli was developed as a counter-selection marker in the most widely used cyanobacterium, Synechocystis sp. PCC 6803. The mazF gene from E. coli was cloned and inserted into a plasmid vector for downstream transformation of Synechocystis. The plasmid construct also contained two homologous flanking regions for integration of the insert into the Synechocystis genome, a nickel-inducible response regulator and promoter to control MazF expression, and a kanamycin resistance gene to serve as the antibiotic marker. In order to ensure the mazF plasmids could be cloned in a MazF-sensitive E. coli host even with slight promoter leakage, MazF expression was toned down by decreasing the efficiency of translation initiation by inserting base pairs between the ribosome binding site and the start codon of the mazF gene. Following successful cloning by E. coli, the mazF plasmids were then used to transform Synechocystis to create mazF mutant strains. Genomic analysis confirmed the successful transformation and segregation of mazF mutant strains containing the desired marker cassette. Phenotypic analysis revealed both growth arrest and production of mazF transcripts in mazF mutant strains following the addition of nickel to the cell cultures, indicating successful nickel-induced MazF expression as desired.
ContributorsNewell, Phoebe Quynh (Co-author) / Newell, Phoebe (Co-author) / Vermaas, Willem (Thesis director) / Wang, Xuan (Committee member) / Li, Shuqin (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
To efficiently produce biofuels and meet the planet’s rising energy demands, different biofuel production methods need to be developed and improved. One of the ways is to produce fatty acid methyl esters (FAMEs) in Synechocystis sp. PCC 6803, a versatile strain of cyanobacteria. In this thesis, Synechocystis was engineered to produce and excrete methyl laurate. In this pathway, first, lauroyl-ACP from fatty acid biosynthesis is converted to laurate by a thioesterase (TE) from Umbellularia californica. Then, the laurate is methylated to methyl laurate by a juvenile hormone acid O-methyltransferase (DmJHAMT) from Drosophila melanogaster. The TE/∆slr1609 strain of Synechocystis sp. PCC 6803 contains the TE gene and lacks the slr1609 gene encoding an acyl–acyl carrier protein synthetase, which functions in free fatty acid reuptake. The DmJHAMT gene was introduced into this strain for FAME production.
The DmJHAMT gene was cloned into a vector that contains neutral sites from the Synechocystis genome, making it suitable for homologous recombination, and a kanamycin resistance gene, for selection. The obtained plasmid was verified using restriction digests and Sanger sequencing. The sequence analysis and comparison of the cDNA in the obtained plasmid and the mRNA transcript of the same gene revealed three amino acid differences. Subsequent comparison with homologous genes in other Drosophila species revealed the differences in the cDNA match those of the other species, and thus, the gene most likely is functional.
The plasmid was transformed into Synechocystis, and PCRs were used to confirm proper integration and segregation. The TE/∆slr1609/DmJHAMT strain produced 62 mg/L methyl laurate in 12 days under a light intensity of 150 µmol photons m-2 s-1, bubbled with 0.5% CO2 at a rate of 30 mL/min, and supplemented with 0.5 mM methionine. The laurate levels did not decrease over time, but instead, remained stagnant after day 3. When the strain was grown in the same conditions without methionine, the laurate concentrations continued to increase above 400 µM, suggesting minimal methyl laurate production and thus a strong need for methionine supplementation. This work provides further evidence of the viability and success of the introduced FAME production pathway, and improved efficiency may be gained in the future.
The DmJHAMT gene was cloned into a vector that contains neutral sites from the Synechocystis genome, making it suitable for homologous recombination, and a kanamycin resistance gene, for selection. The obtained plasmid was verified using restriction digests and Sanger sequencing. The sequence analysis and comparison of the cDNA in the obtained plasmid and the mRNA transcript of the same gene revealed three amino acid differences. Subsequent comparison with homologous genes in other Drosophila species revealed the differences in the cDNA match those of the other species, and thus, the gene most likely is functional.
The plasmid was transformed into Synechocystis, and PCRs were used to confirm proper integration and segregation. The TE/∆slr1609/DmJHAMT strain produced 62 mg/L methyl laurate in 12 days under a light intensity of 150 µmol photons m-2 s-1, bubbled with 0.5% CO2 at a rate of 30 mL/min, and supplemented with 0.5 mM methionine. The laurate levels did not decrease over time, but instead, remained stagnant after day 3. When the strain was grown in the same conditions without methionine, the laurate concentrations continued to increase above 400 µM, suggesting minimal methyl laurate production and thus a strong need for methionine supplementation. This work provides further evidence of the viability and success of the introduced FAME production pathway, and improved efficiency may be gained in the future.
ContributorsSharma, Shuchi (Author) / Vermaas, Willem (Thesis director) / Wang, Xuan (Committee member) / Li, Shuqin (Committee member) / Computer Science and Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05