Searching for interaction partners for the Streptomyces venezuelae FilP protein using a bacterial two-hybrid system
(2018) MOBY01 20181Degree Projects in Molecular Biology
- Abstract
- A system for detecting protein-protein interaction using a bacterial two-hybrid approach, a heterologous expression system in Escherichia coli strain based on adenylate cyclase enzyme reconstitution, was invented twenty years ago (Karimova, et al., 1998, PNAS, 95:5752). Before inventing this system, yeast two-hybrid technology was used for screening putative interaction between either two proteins or DNA-protein in vivo. There are a number of advantages for Bacterial Adenylate Cyclase-Based Two Hybrid (BACTH) system when compared with the yeast two-hybrid method. The BACTH system is a more practical, affordable, easy, and powerful molecular genetics approach for microbiologist with regard to the detection of protein-protein interaction in... (More)
- A system for detecting protein-protein interaction using a bacterial two-hybrid approach, a heterologous expression system in Escherichia coli strain based on adenylate cyclase enzyme reconstitution, was invented twenty years ago (Karimova, et al., 1998, PNAS, 95:5752). Before inventing this system, yeast two-hybrid technology was used for screening putative interaction between either two proteins or DNA-protein in vivo. There are a number of advantages for Bacterial Adenylate Cyclase-Based Two Hybrid (BACTH) system when compared with the yeast two-hybrid method. The BACTH system is a more practical, affordable, easy, and powerful molecular genetics approach for microbiologist with regard to the detection of protein-protein interaction in vivo. The objective of this project was to study putative interactions between two coiled-coil proteins involved in polar growth of Streptomyces bacteria, FilP and DivIVA, using BACTH system. The obtained results showed the FilP self-interaction, DivIVA self-interaction, and FilP-DivIVA interaction in BTH101 E. coli strain. The FilP protein was chosen for screening of genomic libraries to find previously unknown putative interactions of FilP with other proteins). For this, filP gene cloned in the pUT18C, pUT18, pKT25, and pKNT25 plasmids was used as bait to detect putative interactions with unknown proteins cloned in pUT18C-library and pKT25-library. After performing extensive screening of genomic libraries, two genes were identified in more than one clone each, suggesting that they are putative interaction partners for FilP that should be investigated further. These genes are annotated as encoding a cellulose-binding protein consisting mainly of predicted coiled-coil structure, and a Ser or Arg-related nuclear matrix protein. Both genes are broadly conserved in streptomycetes, but their functions have not been investigated. (Less)
- Popular Abstract
- Bacterial two-hybrid system is a fast, cost-effective and popular molecular genetic approach, which used to detect protein-protein interactions in different strains of Escherichia coli bacteria. In this project, using this system helped us to indicate interaction between two different proteins, FilP and DivIVA, in Streptomyces venezuelae bacteria. For this a strain of E. coli and different plasmids carrying separately filP and divIVA genes, were chosen. Then transformation of desired plasmids for detecting FilP self-interaction, DivIVA self-interaction and FilP-DivIVA interaction into strain of E. coli were performed. Obtained results of transformations showed the strong signals of interaction between these proteins.
In the next part of... (More) - Bacterial two-hybrid system is a fast, cost-effective and popular molecular genetic approach, which used to detect protein-protein interactions in different strains of Escherichia coli bacteria. In this project, using this system helped us to indicate interaction between two different proteins, FilP and DivIVA, in Streptomyces venezuelae bacteria. For this a strain of E. coli and different plasmids carrying separately filP and divIVA genes, were chosen. Then transformation of desired plasmids for detecting FilP self-interaction, DivIVA self-interaction and FilP-DivIVA interaction into strain of E. coli were performed. Obtained results of transformations showed the strong signals of interaction between these proteins.
In the next part of the project, screens of genomic libraries to find new putative partner/partners for FilP protein were performed. For this, four plasmids carrying filP gene was selected to indicate interaction between this protein and an unknown library protein/proteins which its gene/genes already were cloned into two different plasmids made by a company. Transformation of desired plasmids into the E. coli strain using bacterial two-hybrid system were performed. Transformants were selected based on ability to grow on lactose and on expression of genes for lactose and maltose catabolism, as these features are used as reporters to indicate positive interaction in this two-hybrid system. Plasmids were extracted from such transformants and their inserts were sequenced and analyzed, leading to identification of two putative partners for FilP protein. These new partners could be used for engineer mutation in S.venezuelae using the λ Red recombination system in E. coli. It is a useful genetic tool could be used to delete specific gene/genes to induce mutation in the desired gene.
Supervisor Klas Flärdh, professor
Molecular Cell Biology, Department of Biology, Lund University
Co-supervisor Markus J. Fröjd, Ph.D. candidate
Molecular Cell Biology, Department of Biology, Lund University (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/8962704
- author
- Karimi, Naghmeh
- supervisor
-
- Klas Flärdh LU
- organization
- course
- MOBY01 20181
- year
- 2018
- type
- M2 - Bachelor Degree
- subject
- language
- English
- id
- 8962704
- date added to LUP
- 2018-10-31 15:07:52
- date last changed
- 2018-10-31 15:07:52
@misc{8962704,
abstract = {{A system for detecting protein-protein interaction using a bacterial two-hybrid approach, a heterologous expression system in Escherichia coli strain based on adenylate cyclase enzyme reconstitution, was invented twenty years ago (Karimova, et al., 1998, PNAS, 95:5752). Before inventing this system, yeast two-hybrid technology was used for screening putative interaction between either two proteins or DNA-protein in vivo. There are a number of advantages for Bacterial Adenylate Cyclase-Based Two Hybrid (BACTH) system when compared with the yeast two-hybrid method. The BACTH system is a more practical, affordable, easy, and powerful molecular genetics approach for microbiologist with regard to the detection of protein-protein interaction in vivo. The objective of this project was to study putative interactions between two coiled-coil proteins involved in polar growth of Streptomyces bacteria, FilP and DivIVA, using BACTH system. The obtained results showed the FilP self-interaction, DivIVA self-interaction, and FilP-DivIVA interaction in BTH101 E. coli strain. The FilP protein was chosen for screening of genomic libraries to find previously unknown putative interactions of FilP with other proteins). For this, filP gene cloned in the pUT18C, pUT18, pKT25, and pKNT25 plasmids was used as bait to detect putative interactions with unknown proteins cloned in pUT18C-library and pKT25-library. After performing extensive screening of genomic libraries, two genes were identified in more than one clone each, suggesting that they are putative interaction partners for FilP that should be investigated further. These genes are annotated as encoding a cellulose-binding protein consisting mainly of predicted coiled-coil structure, and a Ser or Arg-related nuclear matrix protein. Both genes are broadly conserved in streptomycetes, but their functions have not been investigated.}},
author = {{Karimi, Naghmeh}},
language = {{eng}},
note = {{Student Paper}},
title = {{Searching for interaction partners for the Streptomyces venezuelae FilP protein using a bacterial two-hybrid system}},
year = {{2018}},
}