Protein-Protein Interactions Found in Heterocyst Formation of Anabaena Sp. Strain Pcc 7120
Submission Type
Event
Faculty Advisor
Loralyn Cozy
Expected Graduation Date
2018
Location
Center for Natural Sciences, Illinois Wesleyan University
Start Date
4-21-2018 2:00 PM
End Date
4-21-2018 3:00 PM
Disciplines
Education
Abstract
Anabaena sp. strain PCC 7120 is a cyanobacterium that grows in multicellular filaments. Under nitrogen- limiting conditions, Anabaena is capable of forming heterocysts which are differentiated, specialized cells that fix nitrogen for the rest of the cells in the filament. Previous research has shown that HetP is involved in the cessation of the cell cycle and in the formation of heterocysts. However, protein-protein interactions between MinC, MinD, MinE, Ftsz and HetP, have not been previously studied in Anabaena. These interactions can be studied using the bacterial adenylate cyclase two-hybrid system (BACTH). The underlying principle of the BACTH system is the nature of adenylate cyclase found in E.coli. Adenylate cyclase, a protein that produces cAMP can be split into two components, a T25kD half and T18kD half. To assess protein interactions, different respective genes of interest were cloned, in frame, into plasmids containing one of the adenylate cyclase components. This allowed the protein of interest and the complex component to be translated as a fusion protein. These plasmids also contained resistance genes to ampicillin and kanamycin, allowing for the selection of these plasmids in the same cell. When interactions occur between two proteins of interest, the two components of adenylate cyclase come together and produce cAMP. As cAMP levels rise in the cell, cAMP binds to the catabolite activator protein (CAP), a transcription factor, and activates it. When CAP is activated, it binds to the lac operon, resulting in the transcription of β-galactosidase. The production of β-galactosidase was analyzed on antibiotic selective plates containing X-Gal, a compound that turns colonies of E.coli blue was β-galactosidase is present. Using this system, each possible combination of the proteins of interest was assessed by the degree of “blueness” seen on the plates. The degree of “blueness” was compared directly to a positive control, DivIVA, a strain known to create the interaction between the adenylate cyclase halves, and a negative control, which consisted only of empty vectors that did not produce any interaction. Taken together, this revealed the protein-protein interactions within the heterocyst of Anabaena sp. strain PCC 7120.
Protein-Protein Interactions Found in Heterocyst Formation of Anabaena Sp. Strain Pcc 7120
Center for Natural Sciences, Illinois Wesleyan University
Anabaena sp. strain PCC 7120 is a cyanobacterium that grows in multicellular filaments. Under nitrogen- limiting conditions, Anabaena is capable of forming heterocysts which are differentiated, specialized cells that fix nitrogen for the rest of the cells in the filament. Previous research has shown that HetP is involved in the cessation of the cell cycle and in the formation of heterocysts. However, protein-protein interactions between MinC, MinD, MinE, Ftsz and HetP, have not been previously studied in Anabaena. These interactions can be studied using the bacterial adenylate cyclase two-hybrid system (BACTH). The underlying principle of the BACTH system is the nature of adenylate cyclase found in E.coli. Adenylate cyclase, a protein that produces cAMP can be split into two components, a T25kD half and T18kD half. To assess protein interactions, different respective genes of interest were cloned, in frame, into plasmids containing one of the adenylate cyclase components. This allowed the protein of interest and the complex component to be translated as a fusion protein. These plasmids also contained resistance genes to ampicillin and kanamycin, allowing for the selection of these plasmids in the same cell. When interactions occur between two proteins of interest, the two components of adenylate cyclase come together and produce cAMP. As cAMP levels rise in the cell, cAMP binds to the catabolite activator protein (CAP), a transcription factor, and activates it. When CAP is activated, it binds to the lac operon, resulting in the transcription of β-galactosidase. The production of β-galactosidase was analyzed on antibiotic selective plates containing X-Gal, a compound that turns colonies of E.coli blue was β-galactosidase is present. Using this system, each possible combination of the proteins of interest was assessed by the degree of “blueness” seen on the plates. The degree of “blueness” was compared directly to a positive control, DivIVA, a strain known to create the interaction between the adenylate cyclase halves, and a negative control, which consisted only of empty vectors that did not produce any interaction. Taken together, this revealed the protein-protein interactions within the heterocyst of Anabaena sp. strain PCC 7120.