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Development of an expression vector for Caldicellulosiruptor saccharolyticus

Muñoz de las Heras, Alejandro (2012) MOBM19 20112
Degree Projects in Molecular Biology
Abstract
Abstract

To develop a genetic system for the extreme thermophile Caldicellulosirruptor saccharolyticus is the next step in improving hydrogen productivities. Genetic systems are powerful tools to understand the metabolic network of the microorganism and to obtain high hydrogen productivities and yields. The primary aim of this project is to develop an expression vector for C. saccharolyticus. Previous attempts to modify C. saccharolyticus have failed, but new strategies have been developed after the evaluation of the previous works. These new strategies are: to use new plasmids from closely related organisms and application of new adapted transformation and recovery protocols. The plasmids used in this work are native plasmids from C.... (More)
Abstract

To develop a genetic system for the extreme thermophile Caldicellulosirruptor saccharolyticus is the next step in improving hydrogen productivities. Genetic systems are powerful tools to understand the metabolic network of the microorganism and to obtain high hydrogen productivities and yields. The primary aim of this project is to develop an expression vector for C. saccharolyticus. Previous attempts to modify C. saccharolyticus have failed, but new strategies have been developed after the evaluation of the previous works. These new strategies are: to use new plasmids from closely related organisms and application of new adapted transformation and recovery protocols. The plasmids used in this work are native plasmids from C. kristjanssonii (pCALKR01) and C. bescii (pATHE01 and pATHE02). They have been characterized and tested for the compatibility with the intended host, C. saccharolyticus. pCALKR01, in particular, was considered for the construction of the vector. A cassette was constructed containing a kanamycin resistance gene under the promoter from the pyruvate kinase gene from C. saccharolyticus to guarantee the expression in the organism. The cassette was then cloned into pCALKR01. The constructs were then electro-transformed into C. saccharolyticus cells. However, the obtained results could not be verified.

Popular science summary:


Waste products + Bacteria = Hydrogen, the fuel of the future

Hydrogen is presented as a renewable energy currency that can displace the use of petroleum-based fuels. It produces only heat and water upon combustion. Lately more emphasis has been placed in the biohydrogen production by the use of microorganisms via fermentative processes from inexpensive waste feedstocks.

The microorganism used in this work was Caldicellulosiruptor saccharolyticus. It is a strictly anaerobic (it cannot grow in presence of oxygen) and extreme-thermophilic (it grows at 70º C), that possess a natural ability to produce hydrogen and is therefore considered to be a potential candidate for industrial hydrogen production. The primary aim of this project is to improve hydrogen productivities and yields applying genetic engineering by DNA manipulation.

To be able to select those bacteria we have modified the first step in genetic engineering is to introduce a foreign gene that confers a special advantage like resistance to antibiotics. This gene has to be inserted in a circular fragment of DNA called plasmid. The plasmid used is called pCALKR01, coming from C. kristjanssonii. By molecular methods, based upon the DNA sequences of both microorganisms, we have demonstrated that they are evolutionarily closed, making pCALKR01 compatible in C. saccharolyticus.

The Kanamycin resistance gene (from E. coli) confers resistance to this antibiotic with the same name. To activate the gene we also needed to insert a promoter region, which is a DNA sequence that controls the transcription. This promoter is present in the DNA of C. saccharolyticus, so the first study was to determine it (by bioinformatics methods) and then, extract it (by molecular methods).


We have bound both DNA fragments into one (called Cassette KnPr) by a method called Overlap PCR. This cassette was inserted in the plasmid pCALKR01 using a method that uses special enzymes called recombinases. The final construct is called pCALKR01Kn+ expression vector. We also have designed a new protocol to insert this expression vector in C. saccharolyticus.

All of this work is the preparation for having a tool where to insert other genes that are involved in the hydrogen production, among other things. Also is very important to understand how this microorganism works, necessary because it is not well study specie and to manipulate it is required special equipment in the laboratory. It is a step for the production of the energy of the future, the hydrogen.

Advisor: Sudhanshu S. Pawar
Master´s Degree Project 30 credits in 2012
Department of Applied Microbiology, Lund Technical University (Less)
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author
Muñoz de las Heras, Alejandro
supervisor
organization
course
MOBM19 20112
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
3732438
date added to LUP
2013-04-30 12:07:10
date last changed
2013-04-30 12:07:10
@misc{3732438,
  abstract     = {Abstract

To develop a genetic system for the extreme thermophile Caldicellulosirruptor saccharolyticus is the next step in improving hydrogen productivities. Genetic systems are powerful tools to understand the metabolic network of the microorganism and to obtain high hydrogen productivities and yields. The primary aim of this project is to develop an expression vector for C. saccharolyticus. Previous attempts to modify C. saccharolyticus have failed, but new strategies have been developed after the evaluation of the previous works. These new strategies are: to use new plasmids from closely related organisms and application of new adapted transformation and recovery protocols. The plasmids used in this work are native plasmids from C. kristjanssonii (pCALKR01) and C. bescii (pATHE01 and pATHE02). They have been characterized and tested for the compatibility with the intended host, C. saccharolyticus. pCALKR01, in particular, was considered for the construction of the vector. A cassette was constructed containing a kanamycin resistance gene under the promoter from the pyruvate kinase gene from C. saccharolyticus to guarantee the expression in the organism. The cassette was then cloned into pCALKR01. The constructs were then electro-transformed into C. saccharolyticus cells. However, the obtained results could not be verified.

Popular science summary:


Waste products + Bacteria = Hydrogen, the fuel of the future

Hydrogen is presented as a renewable energy currency that can displace the use of petroleum-based fuels. It produces only heat and water upon combustion. Lately more emphasis has been placed in the biohydrogen production by the use of microorganisms via fermentative processes from inexpensive waste feedstocks. 

The microorganism used in this work was Caldicellulosiruptor saccharolyticus. It is a strictly anaerobic (it cannot grow in presence of oxygen) and extreme-thermophilic (it grows at 70º C), that possess a natural ability to produce hydrogen and is therefore considered to be a potential candidate for industrial hydrogen production. The primary aim of this project is to improve hydrogen productivities and yields applying genetic engineering by DNA manipulation.

To be able to select those bacteria we have modified the first step in genetic engineering is to introduce a foreign gene that confers a special advantage like resistance to antibiotics. This gene has to be inserted in a circular fragment of DNA called plasmid. The plasmid used is called pCALKR01, coming from C. kristjanssonii. By molecular methods, based upon the DNA sequences of both microorganisms, we have demonstrated that they are evolutionarily closed, making pCALKR01 compatible in C. saccharolyticus.

The Kanamycin resistance gene (from E. coli) confers resistance to this antibiotic with the same name. To activate the gene we also needed to insert a promoter region, which is a DNA sequence that controls the transcription. This promoter is present in the DNA of C. saccharolyticus, so the first study was to determine it (by bioinformatics methods) and then, extract it (by molecular methods).


We have bound both DNA fragments into one (called Cassette KnPr) by a method called Overlap PCR. This cassette was inserted in the plasmid pCALKR01 using a method that uses special enzymes called recombinases. The final construct is called pCALKR01Kn+ expression vector. We also have designed a new protocol to insert this expression vector in C. saccharolyticus. 

All of this work is the preparation for having a tool where to insert other genes that are involved in the hydrogen production, among other things. Also is very important to understand how this microorganism works, necessary because it is not well study specie and to manipulate it is required special equipment in the laboratory. It is a step for the production of the energy of the future, the hydrogen.

Advisor: Sudhanshu S. Pawar
Master´s Degree Project 30 credits in 2012
Department of Applied Microbiology, Lund Technical University},
  author       = {Muñoz de las Heras, Alejandro},
  language     = {eng},
  note         = {Student Paper},
  title        = {Development of an expression vector for Caldicellulosiruptor saccharolyticus},
  year         = {2012},
}