Projects / Programmes
Bioengineering of adaptive mutation in bacteria Escherichia coli
| Code |
Science |
Field |
Subfield |
| 4.06.00 |
Biotechnical sciences |
Biotechnology |
|
| Code |
Science |
Field |
| T490 |
Technological sciences |
Biotechnology |
adaptive mutation, natural genetic engineering, two-component regulatory system PhoP-PhoQ, magnesium, Ebg operone, mobile genetic elements, insertion sequence IS30, alternative sigma factor RpoS, stationary phase, gene expression, bioengineering, real-time polymerase chain reaction - Q-PCR, quorum sensing inhibitors, biosynthesis optimization, Escherichia coli K-12, neodarvinism, evolution
Researchers (1)
| no. |
Code |
Name and surname |
Research area |
Role |
Period |
No. of publicationsNo. of publications |
| 1. |
21417 |
PhD Rok Krašovec |
Biochemistry and molecular biology |
Head |
2007 - 2009 |
31 |
Organisations (1)
| no. |
Code |
Research organisation |
City |
Registration number |
No. of publicationsNo. of publications |
| 1. |
0431 |
BION Institute |
Ljubljana |
5376041000 |
280 |
Abstract
Adaptive mutation is one of natural genetic engineering systems with which cells’ regulate altering of its own DNA. Because the adaptive mutation is casting doubts in the neodarvinistic evolutionary theory, where the natural selection picks only from random mutations, it represents a true limestone of today’s science. The doctoral dissertation, aimed to study links between the environment and adaptive mutations in bacteria Escherichia coli, proved that environmental magnesium alters the frequency of adaptive mutations. Already in thesis’ discussion we assumed that magnesium is acting differently from the ethidium bromide type of mutagens known to directly damage a DNA sequence. The working hypothesis of the proposed project is worded as follows: environmental magnesium induces adaptive mutations by influencing the activity of the bacterial two-component regulatory system PhoP-PhoQ, otherwise responsible for the magnesium supply in E. coli.
Through here presented project we intend to gather new basic facts that will refute or confirm the working hypothesis bearing for both pure and applied science a very important question. Is there a connection between the PhoP-PhoQ and the mechanism responsible for adaptive mutations? Supposed links between a signal processing system and adaptive mutations will be examined with a modern inexpensive molecular method called a real-time polymerase chain reaction (Q-PCR). With this method we will determine, during the formation of adaptive mutations, a relative expression of four target genes: phoP, phoQ, rpoS and ORF-A (gene that codes for the transposase of the insertion sequence IS30). We will evaluate the effects on the expression from the environmental magnesium, calcium, manganese and iron. Namely, these are elements known to have an impact on the PhoP-PhoQ. Likewise, we intend to determine the expression of four target genes in the environment supplemented with elements that are proven not to affect the PhoP-PhoQ and perform other required positive and negative controls. By performing numerous crosswise experiments we will unequivocally refute or confirm the working hypothesis.
Acquired results will have a high scientific merit, because an understanding of the evolution bears an exceptional importance in life sciences. Namely, mutations are seen as a primary source of biological novelties, but we still do not know much about laws that governs their formation. Experimental methods seeded in the bioengineering are a reliable way of getting an insight into the biological process and here presented project will be one of the first researches where the latest bioengineering knowledge and methodology is going to be used for studying the evolutionary process and mechanisms behind it. At the same time we will make a necessary step ahead in the comprehension of the biological sensing of environmental signals during the starvation, which is very important for an insight of how bacteria behave in natural environments. Also, the collected data will be very useful for the better optimization of biosynthetic processes where very frequently, E. coli is employed. Namely, by using the Q-PCR we will monitor the expression of the gene rpoS that codes a global starvation regulator; a key factor for cells’ differentiation into a more resistant state called a stationary phase. The results will contribute to a better understanding of cell’s regulation of such a mighty important bacterial protein. Research has also a high significance in the applied science, namely, the activity of the quorum sensing system is crucial for inducing the virulence in numerous human, animal and plant’s pathogens. In the light of the alarmingly high microbial resistance, the experimentally proven fact that inhibition of the quorum sensing diminishes microbial virulence sets a quorum sensing inhibition as the most promising antimicrobial strategy of the future with a real chance to become a new leading medical treatment for bacteria
Significance for science
The fact is that bacterial cells sense magnesium with sensory kinase PhoQ and when phoP or PhoQ are knocked out frequency of adaptive mutations is significantly reduced. By supplementing medium with various magnesium concentrations we induce different phoQ-phoP expression rate, but obviously changing the expression rate do not have linear effect on the adaptive mutation process. For mutation inside ebgR apparently the constitutive expression of phoQ/phoP is enough and this is important finding on its own. Exciting and surprising outcome of this project is the cells density effect. Additional experiments are now performed aiming to determine the role of cells density in the adaptive mutation process. We will also consult one of the research groups that have experience in this field. But something is already clear; cells density effect is stable and repeatable and experimental error is surprisingly low. This points out that some rules and regulation must be involved and not only accidental fluctuations. Third important finding is the recognition that adaptive mutation must be understood in terms of complex systemic approach. Namely the complexity theory quite successfully explains the origin of cancer and in my opinion it is also a solid groundwork for understanding adaptive mutation process. All these findings together with the complete argumentation will be very soon send to a respectable international scientific journal.
Significance for the country
The postdoctoral project gave me an opportunity to become aware of many scientific facts that I am now able to integrate in a reasonable whole. The product of this reflection will be an exciting article published in a peer- reviewed scientific journal. The manuscript will encompass all of my past hard work and will point out future prospects for adaptive mutation research. I am now performing experiments on adaptive mutation for almost seven years and I collect so far a wide range of data. I learned a lot in past years and I realize what is to be a head of the research project. I will make use once again all my experiences and social network that I have build to boost scientific progress in my country.
Most important scientific results
Final report,
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Most important socioeconomically and culturally relevant results
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