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Team:CSU Fort Collins/Collab/
From 2014.igem.org
Collaboration with University of Colorado - Boulder
Collaboration is a fundamental need within the scientific community; thus, the Colorado State University and Colorado University at Boulder iGEM teams have collaborated to enhance and support each other’s work.
We tested a part constructed and submitted by the University of Colorado - Boulder. This part, M13ori (for M13 origin of replication) is a non-coding segment of DNA that acts as a packaging signal for M13 phage. Its inclusion onto a plasmid allows for the plasmid’s uptake into a phage capsid. This composite part is called a plasmid. The M13ori does not code for phage coat proteins so a second circular piece of DNA, the helper phagemid, is required to express and assemble the phage coat. This helper phagemid has a disrupted packaging signal so when in the presence of a phagemid, the phagemid will be packaged preferentially. For CU-Boulder we tested the packaging ability of their pSB1C3-M13ori phagemid compared to the M13K07 helper phagemid, which contains kanamycin resistance. We started the phage production protocol on day one then finished the isolation on day two. The phage we isolated were used to infect cells that were then plated onto Chloramphenicol and on kanamycin then the results were analyzed. There was more growth on chloramphenicol than on kanamycin, demonstrating that the M13ori was packaged into and delivered by the M13 phage at a higher rate than M13K07.
Jo from CU iGEM and Krista of CSU iGEM in our lab!
First a cell is transformed with a phagemid and infected with a helper phagmid. The helper phagemid provides the genes necessary for M13 phage production but because its packaging signal is disrupted while the phagemid contains a fully functional packaging signal, the phagemid is uptaken into the assembled phage at a higher rate. However, some phage will still package the helper phagemid. By isolating the phage and infecting a separate stock of cells (1 phage sample, 1 cell sample), the infected cells can be plated under selection to select for cells that were infected by cells containing the phagemid vs the helper phagemid. In this experiment, if the cells grow on chloramphenicol, then they were infected with phage having the pSB1C3-M13ori phagemid; however, cells that grow on kanamycin, were infected with cells having M13K07 helper phagemid. Because the phagemid has the functional packaging signal, it should be packaged more efficiently, and so we expect more colonies to grow on chloramphenicol than kanamycin.
Support collaboration and learn more about CU's project on their 2014 iGEM Wiki.
We tested a part constructed and submitted by the University of Colorado - Boulder. This part, M13ori (for M13 origin of replication) is a non-coding segment of DNA that acts as a packaging signal for M13 phage. Its inclusion onto a plasmid allows for the plasmid’s uptake into a phage capsid. This composite part is called a plasmid. The M13ori does not code for phage coat proteins so a second circular piece of DNA, the helper phagemid, is required to express and assemble the phage coat. This helper phagemid has a disrupted packaging signal so when in the presence of a phagemid, the phagemid will be packaged preferentially. For CU-Boulder we tested the packaging ability of their pSB1C3-M13ori phagemid compared to the M13K07 helper phagemid, which contains kanamycin resistance. We started the phage production protocol on day one then finished the isolation on day two. The phage we isolated were used to infect cells that were then plated onto Chloramphenicol and on kanamycin then the results were analyzed. There was more growth on chloramphenicol than on kanamycin, demonstrating that the M13ori was packaged into and delivered by the M13 phage at a higher rate than M13K07.
Jo from CU iGEM and Krista of CSU iGEM in our lab!
First a cell is transformed with a phagemid and infected with a helper phagmid. The helper phagemid provides the genes necessary for M13 phage production but because its packaging signal is disrupted while the phagemid contains a fully functional packaging signal, the phagemid is uptaken into the assembled phage at a higher rate. However, some phage will still package the helper phagemid. By isolating the phage and infecting a separate stock of cells (1 phage sample, 1 cell sample), the infected cells can be plated under selection to select for cells that were infected by cells containing the phagemid vs the helper phagemid. In this experiment, if the cells grow on chloramphenicol, then they were infected with phage having the pSB1C3-M13ori phagemid; however, cells that grow on kanamycin, were infected with cells having M13K07 helper phagemid. Because the phagemid has the functional packaging signal, it should be packaged more efficiently, and so we expect more colonies to grow on chloramphenicol than kanamycin.
