Difference between revisions of "Part:BBa K2943902:Experience"
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- We included also 3 samples of pure LB in order to use it as blank.
 
- We included also 3 samples of pure LB in order to use it as blank.
  
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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 . For measuring the protein's fluorescence, we used excitation wavelength of 540 nm and emission wavelength of 650 nm.<br>
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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 .  
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Fluorescence was measured using the data below:
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OD: 700nm
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Excitation- Monochromator
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Excitation wavelength: 540nm
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Excitation bandwidth: 50nm
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Emission- Monochromator
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Emission wavelength: 650nm
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Emission bandwidth: 20nm
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Gain: 103 Optimal
  
 
Calculation of the fluorescence intensity:<br> We first measured individual sample intensity as:
 
Calculation of the fluorescence intensity:<br> We first measured individual sample intensity as:

Revision as of 15:20, 20 October 2019


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Applications of BBa_K2943902

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UNIQcfd6e65fe9a20002-partinfo-00000000-QINU UNIQcfd6e65fe9a20002-partinfo-00000001-QINU


BBa_K2943902 TAU_Israel (year 2019)

Part Characterization:
This part is an improved version of an existing part (BBa_K608014). The mutations were detected by using bioinformatics softwares, and then the mutated part was ordered as gblock. Next, we proceeded to wet lab work. We started our lab work by transforming BBa_K608014 from the distribution kit to E. coli DH10beta. Then, after growing starters from the transformation plates overnight, we extracted the plasmids using miniprep.

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.
Fluorescence Experiment:
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: OD: 700nm Excitation- Monochromator Excitation wavelength: 540nm Excitation bandwidth: 50nm Emission- Monochromator Emission wavelength: 650nm Emission bandwidth: 20nm Gain: 103 Optimal

Calculation of the fluorescence intensity:
We first measured individual sample intensity as:

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.


Results:
We received the following fluorescence intensity (Fig.1):
-Non fluorescence bacteria- Mean: 26,766.72; STDEV: 3423.98.
-Original part bacteria- Mean: 57,853.17; STDEV: 2624.75.
-Variation 1 bacteria- Mean: 154,336.4; STDEV: 8994.96.
-Variation 2 bacteria- Mean: 50,705.19; STDEV: 2436.38.

The intensity of our improvment was almost 3 times stronger than the original part.

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