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Team:Alberta/Project
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== '''Overall project''' == | == '''Overall project''' == | ||
| - | + | A microbial color wheel | |
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| + | Summary. We will combine new ideas in regulation with the set of biopigment parts available to produce gradients of color. By using graded regulation, using a new mechanism discovered by our 2010 team, and multiple control elements, we plan to produce a bacterial color wheel with thousands of distinct shades. The developed parts will be useful in biological control circuits, and in providing new flexibility and sensitivity in easy-to-read reporters. | ||
| + | |||
| + | Background and rationale. Flexible and easy-to-use biological reporters are critical for any application in biosensing. Traditional reporters have been limited to a small set of fluorescent proteins and color genes, which produce only all-or-none output. While this is adequate for binary-graded yes/no assays, the limited dynamic range of the output prevents the use of these reporters in contexts calling for a graded response. Moreover, only a single channel of output is available, preventing the application of multiple sensors in a single biological device. There is a clear need for easy-to-use bioreporters capable of graded response and mixed independent output channels. | ||
| + | |||
| + | Objectives. We propose to develop multiple colored outputs, which can be used from a biosensor or circuit, which can be applied together in a multifactorial and graded manner. The outputs will be based on existing genetic parts, such as the Uppsala chromoproteins, and the Cambridge colors. As one demonstration of the system, we will develop a color wheel, using continuously graded control over three pigments matching the three independent colors observable by the human visual system. We will also develop a new, generally useful switching mechanism, based on transcriptional activation competition, discovered by the 2010 iGEM team. Finally, we will continue to advance the assembly methods pioneered by the 2009 and 2010 iGEM teams, while using them in assembly. | ||
| + | |||
| + | Research design and methods. Individual chromoproteins and color generation cassettes will be lifted from parts registry DNA or synthesized. Where useful, basal levels of gene expression will be adjusted using synthetic ribosomal binding sites customized for the protein open reading frames using the Salis Ribosomal Binding Site Calculator (https://salis.psu.edu/software/). The expression range of the bare genes will be assayed when placed behind constitutive promoters of a variety of levels, likely using the Anderson promoter collection. The team will then place the genes under regulatable control, beginning first with the standard lac promoter repressed by LacI under IPTG control, then moving to a new system. We have devised a novel gene control mechanism that switches the activity of common gene control elements from negative control to positive control. The novelty of this method is that any common repressor in E coli can be inverted to an activator, giving it dual function and greatly expanding the application of its control. We will test the dynamic range of the available options, looking for designs which give us the best graded outputs across a suitable range of input substrate concentrations for three separate repressors (cI, LacI and TetR), two of which have small molecule chemical inducers (IPTG for LacI, aTc for TetR). When used in combination, with a lawn of cells poured on top of a dual-gradient plate, multiple colors being expressed in orthogonal gradients will give rise to a “color wheel” of continuous color variation in two dimensions. | ||
| + | |||
| + | Developing and testing this project will require a substantial amount of assembly. Fortunately, we have continued to develop our assembly methods, based on sequential ligation while anchored to a magnetic bead. We propose to use a new system for generating the nonpalindromic sticky ends, based on ligation of short linkers to ends left by common highly efficient cheap restriction enzymes. This avoids the cost and difficulties of Type IIs enzymes such as BsaI. We also will use a new set of ends, selected by an exhaustive algorithm to ensure optimal compatibility, using a rule set based on a set of preliminary experiments. This new end set has more members than the binary A/B system used in 2010 and 2011, which allows more pieces to be assembled per addition step. | ||
| + | |||
| + | Significance and Future Directions. With this project, we hope to achieve both a beautiful color display, which will provide a visually appealing demonstration of the system, and also a useful set of regulatory elements. The applications will be both in education (in the form of the Genomikon kit under development) and in research use, where a set of parts with validated, analog response will be a useful addition to the toolbox of biological circuit design. | ||
== Project Details== | == Project Details== | ||
Revision as of 02:02, 16 July 2012
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Contents |
Overall project
A microbial color wheel
Summary. We will combine new ideas in regulation with the set of biopigment parts available to produce gradients of color. By using graded regulation, using a new mechanism discovered by our 2010 team, and multiple control elements, we plan to produce a bacterial color wheel with thousands of distinct shades. The developed parts will be useful in biological control circuits, and in providing new flexibility and sensitivity in easy-to-read reporters.
Background and rationale. Flexible and easy-to-use biological reporters are critical for any application in biosensing. Traditional reporters have been limited to a small set of fluorescent proteins and color genes, which produce only all-or-none output. While this is adequate for binary-graded yes/no assays, the limited dynamic range of the output prevents the use of these reporters in contexts calling for a graded response. Moreover, only a single channel of output is available, preventing the application of multiple sensors in a single biological device. There is a clear need for easy-to-use bioreporters capable of graded response and mixed independent output channels.
Objectives. We propose to develop multiple colored outputs, which can be used from a biosensor or circuit, which can be applied together in a multifactorial and graded manner. The outputs will be based on existing genetic parts, such as the Uppsala chromoproteins, and the Cambridge colors. As one demonstration of the system, we will develop a color wheel, using continuously graded control over three pigments matching the three independent colors observable by the human visual system. We will also develop a new, generally useful switching mechanism, based on transcriptional activation competition, discovered by the 2010 iGEM team. Finally, we will continue to advance the assembly methods pioneered by the 2009 and 2010 iGEM teams, while using them in assembly.
Research design and methods. Individual chromoproteins and color generation cassettes will be lifted from parts registry DNA or synthesized. Where useful, basal levels of gene expression will be adjusted using synthetic ribosomal binding sites customized for the protein open reading frames using the Salis Ribosomal Binding Site Calculator (https://salis.psu.edu/software/). The expression range of the bare genes will be assayed when placed behind constitutive promoters of a variety of levels, likely using the Anderson promoter collection. The team will then place the genes under regulatable control, beginning first with the standard lac promoter repressed by LacI under IPTG control, then moving to a new system. We have devised a novel gene control mechanism that switches the activity of common gene control elements from negative control to positive control. The novelty of this method is that any common repressor in E coli can be inverted to an activator, giving it dual function and greatly expanding the application of its control. We will test the dynamic range of the available options, looking for designs which give us the best graded outputs across a suitable range of input substrate concentrations for three separate repressors (cI, LacI and TetR), two of which have small molecule chemical inducers (IPTG for LacI, aTc for TetR). When used in combination, with a lawn of cells poured on top of a dual-gradient plate, multiple colors being expressed in orthogonal gradients will give rise to a “color wheel” of continuous color variation in two dimensions.
Developing and testing this project will require a substantial amount of assembly. Fortunately, we have continued to develop our assembly methods, based on sequential ligation while anchored to a magnetic bead. We propose to use a new system for generating the nonpalindromic sticky ends, based on ligation of short linkers to ends left by common highly efficient cheap restriction enzymes. This avoids the cost and difficulties of Type IIs enzymes such as BsaI. We also will use a new set of ends, selected by an exhaustive algorithm to ensure optimal compatibility, using a rule set based on a set of preliminary experiments. This new end set has more members than the binary A/B system used in 2010 and 2011, which allows more pieces to be assembled per addition step.
Significance and Future Directions. With this project, we hope to achieve both a beautiful color display, which will provide a visually appealing demonstration of the system, and also a useful set of regulatory elements. The applications will be both in education (in the form of the Genomikon kit under development) and in research use, where a set of parts with validated, analog response will be a useful addition to the toolbox of biological circuit design.
