Development of a Genetic Engineering Protocol for Caldicellulosiruptor saccharolyticus
(2018) KMBM01 20181Applied Microbiology
Biotechnology
- Abstract
- The genus Caldicellulosiruptor and especially Caldicellulosiruptor saccharolyticus are gaining attention because of their potential as biohydrogen producers from agricultural and forestry waste on an industrial scale. Genetic and in turn metabolic engineering can be used to aid in the characterization of C. saccharolyticus. Additionally, it can be used in strain improvement to overcome limiting factors for the industrial application, such as its low volumetric hydrogen productivity QH2 and osmotolerance. The aim of this thesis is the development of a protocol for the genetic modification of C. saccharolyticus. Consequently, the mutated gene responsible for the uracil auxotrophy of the URA- C. saccharolyticus strain was determined to be... (More)
- The genus Caldicellulosiruptor and especially Caldicellulosiruptor saccharolyticus are gaining attention because of their potential as biohydrogen producers from agricultural and forestry waste on an industrial scale. Genetic and in turn metabolic engineering can be used to aid in the characterization of C. saccharolyticus. Additionally, it can be used in strain improvement to overcome limiting factors for the industrial application, such as its low volumetric hydrogen productivity QH2 and osmotolerance. The aim of this thesis is the development of a protocol for the genetic modification of C. saccharolyticus. Consequently, the mutated gene responsible for the uracil auxotrophy of the URA- C. saccharolyticus strain was determined to be pyrF. This gene would then act as a selection marker conferring uracil prototrophy when inserted into the plasmid vector. Additionally, the vector design included a replication origin for E. coli as well as a putative one with the corresponding replication machinery (Cbes_RS14020) for C. saccharolyticus. From this, a vector plasmid based on pSEVA632 was designed that included the determined selection marker, and replication origin and machinery. Furthermore, the methylase responsible for the unique methylation pattern had to be identified via bioinformatic analysis. Two possible candidates were found, cloned and expressed in the expression host, E. coli BL21 (DE3). The methylases encoded in the loci Csac_2754 and Csac_2755 were then used to modify the plasmid pSEVA632. This methylation was conducted in order to prevent its degradation by the restriction-modification system of C. saccharolyticus but was unsuccessful. Using this protocol, the predicted recognition site GANTC of the mentioned methylases and GASTC of the associated restriction enzyme were proven to be incorrect. In conclusion, a new approach to genetically modify C. saccharolyticus involving the in vitro methylation of DNA was taken. Although promising, this strategy did not succeed but further research in this direction could lead to a viable genetic engineering protocol. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/8948794
- author
- Bostick, James LU
- supervisor
-
- Eoin Byrne LU
- organization
- alternative title
- Utveckling av ett protokoll för genetiskt modifierat Caldicellulosiruptor saccharolyticus
- course
- KMBM01 20181
- year
- 2018
- type
- H2 - Master's Degree (Two Years)
- subject
- keywords
- applied microbiology, teknisk mikrobiologi
- language
- English
- id
- 8948794
- date added to LUP
- 2018-06-18 15:56:56
- date last changed
- 2018-06-18 15:56:56
@misc{8948794,
abstract = {{The genus Caldicellulosiruptor and especially Caldicellulosiruptor saccharolyticus are gaining attention because of their potential as biohydrogen producers from agricultural and forestry waste on an industrial scale. Genetic and in turn metabolic engineering can be used to aid in the characterization of C. saccharolyticus. Additionally, it can be used in strain improvement to overcome limiting factors for the industrial application, such as its low volumetric hydrogen productivity QH2 and osmotolerance. The aim of this thesis is the development of a protocol for the genetic modification of C. saccharolyticus. Consequently, the mutated gene responsible for the uracil auxotrophy of the URA- C. saccharolyticus strain was determined to be pyrF. This gene would then act as a selection marker conferring uracil prototrophy when inserted into the plasmid vector. Additionally, the vector design included a replication origin for E. coli as well as a putative one with the corresponding replication machinery (Cbes_RS14020) for C. saccharolyticus. From this, a vector plasmid based on pSEVA632 was designed that included the determined selection marker, and replication origin and machinery. Furthermore, the methylase responsible for the unique methylation pattern had to be identified via bioinformatic analysis. Two possible candidates were found, cloned and expressed in the expression host, E. coli BL21 (DE3). The methylases encoded in the loci Csac_2754 and Csac_2755 were then used to modify the plasmid pSEVA632. This methylation was conducted in order to prevent its degradation by the restriction-modification system of C. saccharolyticus but was unsuccessful. Using this protocol, the predicted recognition site GANTC of the mentioned methylases and GASTC of the associated restriction enzyme were proven to be incorrect. In conclusion, a new approach to genetically modify C. saccharolyticus involving the in vitro methylation of DNA was taken. Although promising, this strategy did not succeed but further research in this direction could lead to a viable genetic engineering protocol.}},
author = {{Bostick, James}},
language = {{eng}},
note = {{Student Paper}},
title = {{Development of a Genetic Engineering Protocol for Caldicellulosiruptor saccharolyticus}},
year = {{2018}},
}