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Team:Cornell/project/wetlab/fungal toolkit/selectable markers
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Antibiotic resistance is an important component of most molecular cloning; allowing for selection of transformants by killing or inhibiting untransformed or incompletely transformed cells greatly facilitates finding successful transformants. We found three antibiotics that are effective against basidiomycetes and their resistance genes: hygromycin (hph), geneticin (nptII), and phosphinothricin (bar) . We attempted to develop as many configurations of these resistances as possible, putting them behind a variety of promoters, and removing any RFC 10 cut sites that exist in the resistance genes. | Antibiotic resistance is an important component of most molecular cloning; allowing for selection of transformants by killing or inhibiting untransformed or incompletely transformed cells greatly facilitates finding successful transformants. We found three antibiotics that are effective against basidiomycetes and their resistance genes: hygromycin (hph), geneticin (nptII), and phosphinothricin (bar) . We attempted to develop as many configurations of these resistances as possible, putting them behind a variety of promoters, and removing any RFC 10 cut sites that exist in the resistance genes. | ||
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| - | We used the T7 promoter, the promoters for trpC and gpdA in <i>Aspergillus nidulans</i>, and the gpdA promoter from <i>Ganoderma lucidum</i>. For trpC, there is also a terminator, which we appended to PtrpC constructs. The T7 promoter will allow these resistances to be used in orthogonal expression systems, and facilitate transformation of large constructs with several promoters by greatly reducing promoter length. The <i>Aspergillus nidulans</i> promoters should provide high constitutive expression for ascomycetes, and low expression for basidiomycetes, while the <i>Ganoderma lucidum</i> PgpdA provides high constitutive expression for basidiomycetes, and should provide low expression for ascomycetes. | + | <center><img src ="https://static.igem.org/mediawiki/2013/b/ba/Hygromycin_resistance.png"></center><br> |
| - | + | We used the T7 promoter, the promoters for trpC and gpdA in <i>Aspergillus nidulans</i>, and the gpdA promoter from <i>Ganoderma lucidum</i>. For trpC, there is also a terminator, which we appended to PtrpC constructs. The T7 promoter will allow these resistances to be used in orthogonal expression systems, and facilitate transformation of large constructs with several promoters by greatly reducing promoter length. The <i>Aspergillus nidulans</i> promoters should provide high constitutive expression for ascomycetes, and low expression for basidiomycetes, while the <i>Ganoderma lucidum</i> PgpdA provides high constitutive expression for basidiomycetes, and should provide low expression for ascomycetes [1]. | |
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| + | We transformed the construct pC13BV (T7 promoter and hygromycin resistance gene) into the <a href = "https://2013.igem.org/Team:Cornell/project/wetlab/chassis">BL21-A1 strain of <i>E. coli</i></a>, and measured the effects of hygromycin on the wild type and plasmid-containing cells. The diameters of inhibition were statistically significantly different (using a one-tailed student's t-test, with an alpha value of <i>0.005</i>), demonstrating the functionality of our T7 promoter and hygromycin resistance construct. | ||
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| + | We wanted to measure the sensitivity of <i>Ganoderma lucidum</i> to various antibiotics that could potentially be used in its culturing to prevent bacterial contamination. The results we obtained showed insignificant differences in colony size, but interesting slight differences in mycelial growth patterns. The circular layer of mycelium appeared fairly thin in the presence of kanamycin, and of slightly smaller diameter in the presence of chloramphenicol. Ampicillin, on the other hand, seemed to result in a slightly larger diameter of mycelium. The results (after 9 days of growth) are shown in the figure below. | ||
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| + | <h3>References</h3> | ||
| + | 1. Amir Sharon (ed.), <i>Molecular and Cell Biology Methods for Fungi</i>, Methods in Molecular Biology, vol. 638. Turgeon BG, Condon B, Liu J, Zhang N (2010). Protoplast Transformation of Filamentous Fungi. | ||
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Latest revision as of 02:30, 29 October 2013
Selectable Markers
Antibiotic resistance is an important component of most molecular cloning; allowing for selection of transformants by killing or inhibiting untransformed or incompletely transformed cells greatly facilitates finding successful transformants. We found three antibiotics that are effective against basidiomycetes and their resistance genes: hygromycin (hph), geneticin (nptII), and phosphinothricin (bar) . We attempted to develop as many configurations of these resistances as possible, putting them behind a variety of promoters, and removing any RFC 10 cut sites that exist in the resistance genes.
We used the T7 promoter, the promoters for trpC and gpdA in Aspergillus nidulans, and the gpdA promoter from Ganoderma lucidum. For trpC, there is also a terminator, which we appended to PtrpC constructs. The T7 promoter will allow these resistances to be used in orthogonal expression systems, and facilitate transformation of large constructs with several promoters by greatly reducing promoter length. The Aspergillus nidulans promoters should provide high constitutive expression for ascomycetes, and low expression for basidiomycetes, while the Ganoderma lucidum PgpdA provides high constitutive expression for basidiomycetes, and should provide low expression for ascomycetes [1].
