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| − | Template Page | + | InterLab |
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| | ==Overview== | | ==Overview== |
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| − | Fluorescence is a commonly used method for detecting and quantitating gene expression. The InterLab aims to create a more uniform way of measuring fluorescence so that results from different labs can be compared. The results in this study will be collated with all other results for the InterLab study by iGEM HQ. From the results, it is seen that J23101 is the strongest promoter followed by J23106 and J23117 being the weakest, and B0034 is the stronger of the two ribosome binding sites with J364100 being the weaker. Additionally, we chose to investigate how commercial DH5α <i> Esherichia coli </i> derivatives and dimerisation of plasmids can affect gene expression, full methodology and results for this can be seen on the [https://2017.igem.org/Team:Glasgow/Measurement Measurement] page. | + | <b>Fluorescence is a commonly used method for detecting and quantitating gene expression. The InterLab aims to create a more uniform way of measuring fluorescence so that results from different labs can be compared. The results in this study will be collated with all other results for the InterLab study by iGEM HQ. From the results, it is seen that J23101 is the strongest promoter followed by J23106 and J23117 being the weakest, and B0034 is the stronger of the two ribosome binding sites with J364100 being the weaker. Additionally, we chose to investigate how commercial DH5α <i> Esherichia coli </i> derivatives and dimerisation of plasmids can affect gene expression, full methodology and results for this can be seen on the [https://2017.igem.org/Team:Glasgow/Measurement Measurement] page.</b> |
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| | + | ==Introduction== |
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| − | <ref>Kiliç, A. O., Pavlova, S. I., Ma, W. G. & Tao, L. 1996. Analysis of <i>Lactobacillus</i> phages and bacteriocins in American dairy products and characterization of a phage isolated from yogurt. Appl Environ Microbiol, 62, 2111-6.</ref>
| + | The InterLab has been run by iGEM for the past 4 years. It aims to make science more replicable by creating more standardised methods that can be used by any research group with any equipment so that results can be more easily compared. This year the target of the study was to make a more standardised protocol for measuring fluorescence. |
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| | + | For the purpose of the InterLab study, 6 test devices were used. Each device contained one of three different promoters, and one of two different ribosome binding sites upstream of a gene for green fluorescent protein (E0040) and two terminators (B0010 and B0012). The promoters used in the study were: J23101 (strong); J23106 (medium); and J23117 (weak). The ribosome binding sites (RBS) used were; B0034; and J364100 (an element designed by Mutalik et al <ref>Mutalik, V., Guimaraes, J., Cambray, G., Lam, C., Christoffersen, M., Mai, Q., Tran, A., Paull, M., Keasling, J., Arkin, A. and Endy, D. (2013). Precise and reliable gene expression via standard transcription and translation initiation elements. <i>Nature Methods</i>, 10(4), pp.354-360. </ref>). The relative strengths of these two ribosome binding sites were not known at the start of this study. Diagrams showing the constituent parts of each test device can be seen in figure 1. Along with these six devices, were a positive and negative control, the positive being I20270 – contains the J23151 promoter, B0032 ribosome binding site, E0040 gfp gene, B0010 terminator and the B0012 terminator- and R0040 –tetR repressible promotor with no gfp gene, which will not express GFP-. All devices used for testing were in the pSB1C3 plasmid and transformed into DH5α <i>E. coli</i>. |
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| − | ==Aims==
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| − | *Aim 1
| + | ==Materials and Methods== |
| − | **Sub-aim 1
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| − | **Sub-aim 2
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| − | *Aim 2
| + | ====Transformations==== |
| − | **Sub-aim 1
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| − | **Sub-aim 2
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| | + | These were carried out as per our [https://2017.igem.org/Team:Glasgow/Protocols standard calcium chloride protocol.] |
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| − | ==Materials and Methods==
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| − | ===Condition set up=== | + | ====Calibration==== |
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| | + | Both the OD600 reference point and the fluorescein fluorescence standard curve were carried out as per the [https://static.igem.org/mediawiki/2017/8/85/InterLab_2017_Plate_Reader_Protocol.pdf InterLab 2017 Plate Reader Protocol] |
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| − | ===Sample preparation===
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| − | <small>
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| − | *1
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| − | *2
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| − | *3
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| − | </small>
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| − | {{GlasgowWikiImage|image=Glasgow2017_caption_image1.JPG|caption=<b>Table 1:</b> Optical density analysis of <i>S. thermophilus</i> growth}}
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Overview
Fluorescence is a commonly used method for detecting and quantitating gene expression. The InterLab aims to create a more uniform way of measuring fluorescence so that results from different labs can be compared. The results in this study will be collated with all other results for the InterLab study by iGEM HQ. From the results, it is seen that J23101 is the strongest promoter followed by J23106 and J23117 being the weakest, and B0034 is the stronger of the two ribosome binding sites with J364100 being the weaker. Additionally, we chose to investigate how commercial DH5α Esherichia coli derivatives and dimerisation of plasmids can affect gene expression, full methodology and results for this can be seen on the Measurement page.
Introduction
The InterLab has been run by iGEM for the past 4 years. It aims to make science more replicable by creating more standardised methods that can be used by any research group with any equipment so that results can be more easily compared. This year the target of the study was to make a more standardised protocol for measuring fluorescence.
For the purpose of the InterLab study, 6 test devices were used. Each device contained one of three different promoters, and one of two different ribosome binding sites upstream of a gene for green fluorescent protein (E0040) and two terminators (B0010 and B0012). The promoters used in the study were: J23101 (strong); J23106 (medium); and J23117 (weak). The ribosome binding sites (RBS) used were; B0034; and J364100 (an element designed by Mutalik et al [1]). The relative strengths of these two ribosome binding sites were not known at the start of this study. Diagrams showing the constituent parts of each test device can be seen in figure 1. Along with these six devices, were a positive and negative control, the positive being I20270 – contains the J23151 promoter, B0032 ribosome binding site, E0040 gfp gene, B0010 terminator and the B0012 terminator- and R0040 –tetR repressible promotor with no gfp gene, which will not express GFP-. All devices used for testing were in the pSB1C3 plasmid and transformed into DH5α E. coli.
Materials and Methods
Transformations
These were carried out as per our standard calcium chloride protocol.
Calibration
Both the OD600 reference point and the fluorescein fluorescence standard curve were carried out as per the InterLab 2017 Plate Reader Protocol
Results
Discussion
References
- ↑ Mutalik, V., Guimaraes, J., Cambray, G., Lam, C., Christoffersen, M., Mai, Q., Tran, A., Paull, M., Keasling, J., Arkin, A. and Endy, D. (2013). Precise and reliable gene expression via standard transcription and translation initiation elements. Nature Methods, 10(4), pp.354-360.
