Difference between revisions of "Team:Aalto-Helsinki/InterLab"
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Measurements should be able to repeat, but because every laboratory has their own practices, intruments and devices, it is sometimes challenging and results are not same even though the protocol would be. This year, iGEM teams measured absorbance and fluorescence. E. Coli was transformed with different green fluorescence protein plasmids and plate reader was used to measure the values. | Measurements should be able to repeat, but because every laboratory has their own practices, intruments and devices, it is sometimes challenging and results are not same even though the protocol would be. This year, iGEM teams measured absorbance and fluorescence. E. Coli was transformed with different green fluorescence protein plasmids and plate reader was used to measure the values. | ||
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| − | < | + | <font face="calibri" size="5">Results</font> |
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| − | < | + | <font face="calibri" size="4">Competency</font> |
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Cells were made competent by using iGEM protocol [1]. LB was used as a medium instead of SOB or SOC, which may have affected on the competence. Cell competency was measured by using Competent Cell Test Kit provided by iGEM. The kit contained two vials of purified plasmid DNA from BBa_J04450 in plasmid ackbone pSB1C3. The concentrations of the vials were 10 pg/µl and 100 pg/µl. Measurements were done according to iGEM protocol [1]. Escherichia coli DH5α cells were used in the experiment. According to the iGEM protocol, competent cells should have an efficiency of 1.5 ⋅ 10<sup>8</sup> to 6 ⋅ 10<sup>8</sup> cfu/µg. The transformation efficiency was 6.6 ⋅ 10<sup>5</sup> cfu/µg, which is very low compared to that. However, the measurements were able to accomplish. | Cells were made competent by using iGEM protocol [1]. LB was used as a medium instead of SOB or SOC, which may have affected on the competence. Cell competency was measured by using Competent Cell Test Kit provided by iGEM. The kit contained two vials of purified plasmid DNA from BBa_J04450 in plasmid ackbone pSB1C3. The concentrations of the vials were 10 pg/µl and 100 pg/µl. Measurements were done according to iGEM protocol [1]. Escherichia coli DH5α cells were used in the experiment. According to the iGEM protocol, competent cells should have an efficiency of 1.5 ⋅ 10<sup>8</sup> to 6 ⋅ 10<sup>8</sup> cfu/µg. The transformation efficiency was 6.6 ⋅ 10<sup>5</sup> cfu/µg, which is very low compared to that. However, the measurements were able to accomplish. | ||
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The transformation of E. Coli DH5α was performed with six different plasmids and negative and positive control. The plasmids of test devices were GFP that had small differences in the structure, e.g., different promoter. | The transformation of E. Coli DH5α was performed with six different plasmids and negative and positive control. The plasmids of test devices were GFP that had small differences in the structure, e.g., different promoter. | ||
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Test Device 5 BBa_J364008 | Test Device 5 BBa_J364008 | ||
Test Device 6 BBa_J364009 | Test Device 6 BBa_J364009 | ||
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| − | < | + | <font face="calibri" size="4">OD<sub>600</sub> Reference Point</font> |
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Because plate reader measures absorbance that depends on volume, it was necessary to measure the ratio of OD600 to Abs600. LUDOX CL-X and sterile water were used in the experiment. LUDOX CL-X is a 45 % colloidal silica suspension. Table 3 shows the results. | Because plate reader measures absorbance that depends on volume, it was necessary to measure the ratio of OD600 to Abs600. LUDOX CL-X and sterile water were used in the experiment. LUDOX CL-X is a 45 % colloidal silica suspension. Table 3 shows the results. | ||
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| − | < | + | <font face="calibri" size="4">Particle Standard Curve</font> |
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Microsphere suspension was used to construct the particle standard curve. Microspheres are the same size than cells and the optical properties are also similar with the cells that were used. Microspheres were diluted in order to get the relation between absorbance and the amount of cells. The results were used to estimate the amount of cells in certain absorbances. Figures 5 and 6 show the results. | Microsphere suspension was used to construct the particle standard curve. Microspheres are the same size than cells and the optical properties are also similar with the cells that were used. Microspheres were diluted in order to get the relation between absorbance and the amount of cells. The results were used to estimate the amount of cells in certain absorbances. Figures 5 and 6 show the results. | ||
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The fluorescence values vary depending on the device. Fluorescence standard curve makes it possible to compare fluorescence measurements between teams. Fluorescein was serially diluted in phosphate buffered saline in order to get different fluorescein concentration. Finally, fluorescence was measured by using plate reader. Figures 3 and 4 show the results. | The fluorescence values vary depending on the device. Fluorescence standard curve makes it possible to compare fluorescence measurements between teams. Fluorescein was serially diluted in phosphate buffered saline in order to get different fluorescein concentration. Finally, fluorescence was measured by using plate reader. Figures 3 and 4 show the results. | ||
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Transformation was done according to the iGEM protocol [3]. After that, two colonies were picked from each plate and inoculated in LB medium, which contained chroramphenicol. Cells were incubated over night and samples were taken in the next morning and six hours after that. Plate reader protocol [4] has more detailed description about the process. | Transformation was done according to the iGEM protocol [3]. After that, two colonies were picked from each plate and inoculated in LB medium, which contained chroramphenicol. Cells were incubated over night and samples were taken in the next morning and six hours after that. Plate reader protocol [4] has more detailed description about the process. | ||
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| − | < | + | <font face="calibri" size="3">Absorbance</font> |
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| − | < | + | <font face="calibri" size="3">Fluorescence</font> |
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[1] http://parts.igem.org/Help:Protocols/Competent_Cells <br> | [1] http://parts.igem.org/Help:Protocols/Competent_Cells <br> | ||
Revision as of 08:05, 20 July 2018
Introduction
Measurements should be able to repeat, but because every laboratory has their own practices, intruments and devices, it is sometimes challenging and results are not same even though the protocol would be. This year, iGEM teams measured absorbance and fluorescence. E. Coli was transformed with different green fluorescence protein plasmids and plate reader was used to measure the values.
Results
Competency
Cells were made competent by using iGEM protocol [1]. LB was used as a medium instead of SOB or SOC, which may have affected on the competence. Cell competency was measured by using Competent Cell Test Kit provided by iGEM. The kit contained two vials of purified plasmid DNA from BBa_J04450 in plasmid ackbone pSB1C3. The concentrations of the vials were 10 pg/µl and 100 pg/µl. Measurements were done according to iGEM protocol [1]. Escherichia coli DH5α cells were used in the experiment. According to the iGEM protocol, competent cells should have an efficiency of 1.5 ⋅ 108 to 6 ⋅ 108 cfu/µg. The transformation efficiency was 6.6 ⋅ 105 cfu/µg, which is very low compared to that. However, the measurements were able to accomplish.
Parts
The transformation of E. Coli DH5α was performed with six different plasmids and negative and positive control. The plasmids of test devices were GFP that had small differences in the structure, e.g., different promoter. Device Part Number Negative control BBa_R0040 Positive control BBa_I20270 Test Device 1 BBa_J364000 Test Device 2 BBa_J364001 Test Device 3 BBa_J364002 Test Device 4 BBa_J364007 Test Device 5 BBa_J364008 Test Device 6 BBa_J364009
OD600 Reference Point
Because plate reader measures absorbance that depends on volume, it was necessary to measure the ratio of OD600 to Abs600. LUDOX CL-X and sterile water were used in the experiment. LUDOX CL-X is a 45 % colloidal silica suspension. Table 3 shows the results.
Particle Standard Curve
Microsphere suspension was used to construct the particle standard curve. Microspheres are the same size than cells and the optical properties are also similar with the cells that were used. Microspheres were diluted in order to get the relation between absorbance and the amount of cells. The results were used to estimate the amount of cells in certain absorbances. Figures 5 and 6 show the results.
Fluorescence Standard Curve
The fluorescence values vary depending on the device. Fluorescence standard curve makes it possible to compare fluorescence measurements between teams. Fluorescein was serially diluted in phosphate buffered saline in order to get different fluorescein concentration. Finally, fluorescence was measured by using plate reader. Figures 3 and 4 show the results.
Cell Measurements
Transformation was done according to the iGEM protocol [3]. After that, two colonies were picked from each plate and inoculated in LB medium, which contained chroramphenicol. Cells were incubated over night and samples were taken in the next morning and six hours after that. Plate reader protocol [4] has more detailed description about the process.
Absorbance
Fluorescence
[1] http://parts.igem.org/Help:Protocols/Competent_Cells
[2] http://parts.igem.org/Help:2017_Competent_Cell_Test_Kit
[3] http://parts.igem.org/Help:Protocols/Transformation
[4] https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf
Measurements should be able to repeat, but because every laboratory has their own practices, intruments and devices, it is sometimes challenging and results are not same even though the protocol would be. This year, iGEM teams measured absorbance and fluorescence. E. Coli was transformed with different green fluorescence protein plasmids and plate reader was used to measure the values.
Results
Competency
Cells were made competent by using iGEM protocol [1]. LB was used as a medium instead of SOB or SOC, which may have affected on the competence. Cell competency was measured by using Competent Cell Test Kit provided by iGEM. The kit contained two vials of purified plasmid DNA from BBa_J04450 in plasmid ackbone pSB1C3. The concentrations of the vials were 10 pg/µl and 100 pg/µl. Measurements were done according to iGEM protocol [1]. Escherichia coli DH5α cells were used in the experiment. According to the iGEM protocol, competent cells should have an efficiency of 1.5 ⋅ 108 to 6 ⋅ 108 cfu/µg. The transformation efficiency was 6.6 ⋅ 105 cfu/µg, which is very low compared to that. However, the measurements were able to accomplish.
Parts
The transformation of E. Coli DH5α was performed with six different plasmids and negative and positive control. The plasmids of test devices were GFP that had small differences in the structure, e.g., different promoter. Device Part Number Negative control BBa_R0040 Positive control BBa_I20270 Test Device 1 BBa_J364000 Test Device 2 BBa_J364001 Test Device 3 BBa_J364002 Test Device 4 BBa_J364007 Test Device 5 BBa_J364008 Test Device 6 BBa_J364009
OD600 Reference Point
Because plate reader measures absorbance that depends on volume, it was necessary to measure the ratio of OD600 to Abs600. LUDOX CL-X and sterile water were used in the experiment. LUDOX CL-X is a 45 % colloidal silica suspension. Table 3 shows the results.
Particle Standard Curve
Microsphere suspension was used to construct the particle standard curve. Microspheres are the same size than cells and the optical properties are also similar with the cells that were used. Microspheres were diluted in order to get the relation between absorbance and the amount of cells. The results were used to estimate the amount of cells in certain absorbances. Figures 5 and 6 show the results.
Fluorescence Standard Curve
The fluorescence values vary depending on the device. Fluorescence standard curve makes it possible to compare fluorescence measurements between teams. Fluorescein was serially diluted in phosphate buffered saline in order to get different fluorescein concentration. Finally, fluorescence was measured by using plate reader. Figures 3 and 4 show the results.
Cell Measurements
Transformation was done according to the iGEM protocol [3]. After that, two colonies were picked from each plate and inoculated in LB medium, which contained chroramphenicol. Cells were incubated over night and samples were taken in the next morning and six hours after that. Plate reader protocol [4] has more detailed description about the process.
Absorbance
Fluorescence
[1] http://parts.igem.org/Help:Protocols/Competent_Cells
[2] http://parts.igem.org/Help:2017_Competent_Cell_Test_Kit
[3] http://parts.igem.org/Help:Protocols/Transformation
[4] https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf