Difference between revisions of "Part:BBa K2943902:Experience"
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<partinfo>BBa_K2943902</partinfo> | <partinfo>BBa_K2943902</partinfo> | ||
− | <I>TAU_Israel</I> | + | <I>TAU_Israel (year 2019)</I> |
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<b>Part Characterization:</b><br> | <b>Part Characterization:</b><br> | ||
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Using PCR reaction, we amplified part of the plasmid, excluding part of the mRFP and RBS. This enabled us to insert the gblocks into the plasmid which included already the appropriate backbone and overlaps for the assembly. | Using PCR reaction, we amplified part of the plasmid, excluding part of the mRFP and RBS. This enabled us to insert the gblocks into the plasmid which included already the appropriate backbone and overlaps for the assembly. | ||
− | The gblock was inserted using Gibson Assembly. Before the assembly, we have done DPN1 to ensure no unwanted DNA template was present and used PCR clean kit to prepare the amplified backbone. Then we proceeded to the Gibson reaction and transformed the resulting plasmid into <em> E. coli</em> DH10beta. | + | The gblock was inserted using Gibson Assembly. Before the assembly, we have done DPN1 to ensure no unwanted DNA template was present and used PCR clean kit to prepare the amplified backbone. Then we proceeded to the Gibson reaction and transformed the resulting plasmid into <em> E. coli</em> DH10beta. <br> |
+ | <b>Fluorescence Experiment:</b><br> | ||
+ | We have used plate reader machine. The plate contained 3 samples of each of the next bacteria: | ||
+ | |||
+ | -DH10beta containing non fluorescent plasmid for negative control. | ||
+ | |||
+ | -DH10beta containing the original part. | ||
+ | |||
+ | -DH10beta containing this part. | ||
+ | |||
+ | - We included also 3 samples of pure LB in order to use it as blank. | ||
+ | |||
+ | Because RFP can absorb light at 600nm and lead to false measurements, concentration of bacteria was estimated using OD700 as recommended previously by the iGEM committee .Fluorescence was measured using the data below:<br> | ||
+ | OD: 700nm<br> | ||
+ | Excitation- Monochromator<br> | ||
+ | Excitation wavelength: 540nm<br> | ||
+ | Excitation bandwidth: 50nm<br> | ||
+ | Emission- Monochromator<br> | ||
+ | Emission wavelength: 650nm<br> | ||
+ | Emission bandwidth: 20nm<br> | ||
+ | Gain: 103 Optimal<br> | ||
+ | |||
+ | <b>Calculation of the fluorescence intensity:</b><br> We first measured individual sample intensity as: | ||
+ | |||
+ | Individual Intensity= (Florescence of plasmid - florescence of LB) / # Colonies in OD700<br> | ||
+ | |||
+ | Then, for each type of bacteria, we calculated the average of the three appropriate samples intensity.<br> | ||
+ | |||
+ | |||
+ | <b>Results:</b><br> | ||
+ | We received the following fluorescence intensity (Fig.1):<br> | ||
+ | -Non fluorescence bacteria- Mean: 26,766.72; STDEV: 3423.98.<br> | ||
+ | -Original part bacteria- Mean: 57,853.17; STDEV: 2624.75.<br> | ||
+ | -Variation 1 bacteria- Mean: 154,336.4; STDEV: 8994.96.<br> | ||
+ | -Variation 2 bacteria- Mean: 50,705.19; STDEV: 2436.38.<br> | ||
+ | |||
+ | The intensity of our improved part was almost 3 times stronger than the original part. | ||
|}; | |}; |
Latest revision as of 15:26, 20 October 2019
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BBa_K2943902 TAU_Israel (year 2019) |
Part Characterization: Using PCR reaction, we amplified part of the plasmid, excluding part of the mRFP and RBS. This enabled us to insert the gblocks into the plasmid which included already the appropriate backbone and overlaps for the assembly.
The gblock was inserted using Gibson Assembly. Before the assembly, we have done DPN1 to ensure no unwanted DNA template was present and used PCR clean kit to prepare the amplified backbone. Then we proceeded to the Gibson reaction and transformed the resulting plasmid into E. coli DH10beta. -DH10beta containing non fluorescent plasmid for negative control. -DH10beta containing the original part. -DH10beta containing this part. - We included also 3 samples of pure LB in order to use it as blank. Because RFP can absorb light at 600nm and lead to false measurements, concentration of bacteria was estimated using OD700 as recommended previously by the iGEM committee .Fluorescence was measured using the data below: Calculation of the fluorescence intensity: Individual Intensity= (Florescence of plasmid - florescence of LB) / # Colonies in OD700 Then, for each type of bacteria, we calculated the average of the three appropriate samples intensity.
The intensity of our improved part was almost 3 times stronger than the original part. |