Difference between revisions of "Part:BBa K2943902:Experience"

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:<vbr> 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.