Termofilinių bakterijų genetinė transformacija /

Thermophilic bacteria are considered to be useful biotechnological objects, since this type of bacteria has a lot of possible uses in various industrial fields, where these microorganisms serve as a source of many thermostable enzymes as well as biotechnologically produced metabolites. However, due to the lack of reliable genetic transformation systems for thermophiles, genetic manipulation and industrial application remains a challenging approach. Thus, further studies on thermophilic bacteria transformation are highly required. The aim of this research was to transform three thermophilic bacteria strains – Parageobacillus toebii DSM14950T, Geobacillus stearothermophilus 15 ir Aeribacillus pallidus 8. Development of transformation system for the latter two strains was based on in vivo methylation by expressing methyltransferase genes in Escherichia coli strains in order to avoid restriction of plasmids by the host R-M systems. Using the REBASE database, several types of R-M systems were identified in A. pallidus 8 and G. stearothermophilus 15 strains. Only two of A. pallidus 8 R-M system genes, RM1.2 and RM2.1, were successfully transferred to the E. coli DH5α and E. coli GM119 strains. After the gene induction and plasmid extraction, none of the R-M genes containing vectors were transmitted to the final host of A. pallidus 8. During the investigation of G. stearothermophilus 15 R-M systems, E. coli GM119 bacteria was transformed with four methyltransferase genes – RM2, RM3.1, RM3.4 ir RM3.5 – yet only RM2 and RM3.4 were transferred to E. coli DH5α. Transformation of G. stearothermophilus 15 with the extracted plasmids was not achieved. Another goal involved the evaluation of P. toebii DSM14950T, G. stearothermophilus 15 and A. pallidus 8 transformation efficiency dependence on Dam and Dcm methylation as well as different replication initiation proteins. After the transfer of methylated and non-methylated plasmids containing various replication initiation proteins, no colonies were obtained indicating these factors do not have an impact on transformation efficiency of the three thermophilic strains.

Grantor dc:publisher

Institutional Repository of Vilnius University

Year dc:date

2020

Author dc:creator

• Kondrataitė, Aušra,

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

lit

Repository record dc:identifier

https://repository.vu.lt/VU:ELABAETD193302572&prefLang=en_US

OAI identifier oai:identifier

oai:vu.lt:elaba:193302572