Presentation Information
[P04-575]Improvement of the transformation efficiency of the marine diatom via bacterial conjugation using native sequences
○Hyun-Sik Yun1, Iwane Suzuki1, Yoshiaki Maeda1 (1. University of Tsukuba (Japan))
Keywords:
Episome,Conjugation,Marine diatom,Phaeodactylum tricornutum,Centromeres (CENs),Autonomous replication sequences (ARSs)
[Objectives]
Microalgae, as autotrophic organisms, possess a unique appeal as a bioresource that distinguishes them from other heterotrophic microorganisms. Nevertheless, a genetic engineering approach is necessary to enhance the value of microalgae. The introduction of episomes was proposed as a more innovative methodology, and yeast centromeres (CENs)/native autonomous replication sequences (ARSs) were applied to enable the maintenance of episomes within microalgal cells. However, it faces the challenge of needing to improve the problem that the number of episome copies is less than two, and there are limitations to the expression level of the introduced gene in the transformant. To improve this limitation, we introduced and evaluated native sequences expected to function as CEN/ARS in an episome for Phaeodactylum tricornutum, one of the model microalgae species.
[Method]
Conjugation was applied to introduce the circular plasmid as an episome. The episome was designed with the pPtPuc3 plasmid as the backbone, and cassettes for the expression of the bleomycin-resistance gene and enhanced green fluorescent protein (EGFP), as well as sequences of native CEN and ARS, were added. The designed cargo plasmid was introduced into P. tricornutum via Escherichia coli (with the helper plasmid pTA-Mob2). The efficiency of transformant production was evaluated, and EGFP expression was confirmed using microscopy and Western blot. To confirm that the introduced plasmid exists as an episome, PCR and plasmid extraction were performed.
[Results & Discussion]
As a result of comparing the frequency of transformant production via conjugation, the plasmid with added native CEN and ARS (0.24 ± 0.37 colonies/106 cells, max: 1.82, n=150) was superior to the plasmid with added yeast CEN/ARS (0.03 ± 0.07 colonies/106 cells, max: 0.31, n=150). Previous studies have reported that episome deletions occur as a result of introduction via conjugation, and the same phenomenon was confirmed at a higher frequency in results for native CEN and ARS (Insert: 41.67%, Backbone: 8.33%) compared to yeast CEN/ARS (Insert: 11.11%, Backbone: 9.62%). Transformants without a fluorescence signal were observed due to deletions in regions related to EGFP expression, and cases were confirmed where the extracted plasmid could not be retransformed into E. coli due to deletions in the backbone region. While the efficiency of transformant production can be improved, a solution is needed for issues related to deletions.
[Conclusion]Our study suggests that plasmid design utilizing native sequences can maximize the transformation efficiency. Furthermore, we propose that the deletion problem must be resolved in the production of microalgal transformants through conjugation. Our future study will focus on analyzing deletion patterns and identifying mechanisms to enable the introduction of stable episomes.
Microalgae, as autotrophic organisms, possess a unique appeal as a bioresource that distinguishes them from other heterotrophic microorganisms. Nevertheless, a genetic engineering approach is necessary to enhance the value of microalgae. The introduction of episomes was proposed as a more innovative methodology, and yeast centromeres (CENs)/native autonomous replication sequences (ARSs) were applied to enable the maintenance of episomes within microalgal cells. However, it faces the challenge of needing to improve the problem that the number of episome copies is less than two, and there are limitations to the expression level of the introduced gene in the transformant. To improve this limitation, we introduced and evaluated native sequences expected to function as CEN/ARS in an episome for Phaeodactylum tricornutum, one of the model microalgae species.
[Method]
Conjugation was applied to introduce the circular plasmid as an episome. The episome was designed with the pPtPuc3 plasmid as the backbone, and cassettes for the expression of the bleomycin-resistance gene and enhanced green fluorescent protein (EGFP), as well as sequences of native CEN and ARS, were added. The designed cargo plasmid was introduced into P. tricornutum via Escherichia coli (with the helper plasmid pTA-Mob2). The efficiency of transformant production was evaluated, and EGFP expression was confirmed using microscopy and Western blot. To confirm that the introduced plasmid exists as an episome, PCR and plasmid extraction were performed.
[Results & Discussion]
As a result of comparing the frequency of transformant production via conjugation, the plasmid with added native CEN and ARS (0.24 ± 0.37 colonies/106 cells, max: 1.82, n=150) was superior to the plasmid with added yeast CEN/ARS (0.03 ± 0.07 colonies/106 cells, max: 0.31, n=150). Previous studies have reported that episome deletions occur as a result of introduction via conjugation, and the same phenomenon was confirmed at a higher frequency in results for native CEN and ARS (Insert: 41.67%, Backbone: 8.33%) compared to yeast CEN/ARS (Insert: 11.11%, Backbone: 9.62%). Transformants without a fluorescence signal were observed due to deletions in regions related to EGFP expression, and cases were confirmed where the extracted plasmid could not be retransformed into E. coli due to deletions in the backbone region. While the efficiency of transformant production can be improved, a solution is needed for issues related to deletions.
[Conclusion]Our study suggests that plasmid design utilizing native sequences can maximize the transformation efficiency. Furthermore, we propose that the deletion problem must be resolved in the production of microalgal transformants through conjugation. Our future study will focus on analyzing deletion patterns and identifying mechanisms to enable the introduction of stable episomes.
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