Part:BBa_K608014
strong promoter and medium RBS with RFP
This part consists of a strong promoter with medium RBS (PR2) and tagged with RFP to quantify the expression.
The RFP fluorescence was measured with a plate reader:
The fluorescence intensity and protein concentration were measured with the FLUOstar Omega,
which is a multi-mode microplate reader.
Samples were pipetted into the microplate and analyzed via the plate reader. In this experiment we focused on the protein concentration and the fluorescence intensity of RFP.
We measured the protein concentration with the bradford-assay. This is a method to determine the total protein
concentration. To analyze the protein concentration of the samples, Coomassie Brillant Blue was pippeted to each sample. With the binding of the dye to the proteins the color changes from dark red to blue. The more protein in the solution the more Coomassie dye can bind to proteins and the more the color changes into blue. The absorption of bound Coomassie dye is 595nm. The absorbance is proportional with the amount of bound dye. With a series of Bovine Serum Albumin (BSA) measurements the exact protein concentration of the samples can be determined. BSA acts like a “marker†because the concentration of BSA is known and with a linear calibration line the exact protein concentration can be detected.
With RFP (Red Fluorescence Protein) the activity of promoter and RBS can be quantified.
RFP served like GFP as a reporter to determine the expression level. RFP is excited at a wavelength of 580nm. The more RFP in the solution the more is the RFP fluorescence intensity.The plate reader illuminates the samples with a high energy xenon flash lamp. Optical filters or monochromator create the exact wavelength. The more RFP in the sample the higher is the RFP fluorescence intensity. The intensity is collected with the second optical system and is detected with a side window photomultiplier tube.
Promoter and RBS:
PR1: strong Promoter (J23104) strong RBS (B0034)
PR2: strong Promoter (J23104) medium RBS (B0032)
PR3: strong Promoter (J23104) weak RBS (B0031)
PR4: medium Promoter (J23110) strong RBS (B0034)
PR5: medium Promoter (J23110) medium RBS (B0032)
PR6: medium Promoter (J23110) weak RBS (B0031)
| sample | PR1 | PR2 | PR4 | PR5 | PR6 |
| RFP fluorescence intensity | 25310.5 | 27144.5 | 9675.1 | 13415.9 | 685.6 |
| factor | 36.9 | 39.6 | 14.1 | 19.6 | 1.0 |
The results of this test show that PR2 is 39,9 times stronger than PR6. Although the PR1 should have the strongest expression of RFP, in this case PR2 had the strongest expression of RFP. The fluorescence intensity of RFP varies, and because of lack of time we could not repeat this experiment. We have also tested the promoter and RBS activity with GFP as a reporter and the results deviate from this experiment. So we are looking forward to test this system another time.
Contributions:
Group: TAU_Israel (Year 2019)
Author: May_1
Summary: Improved the expression of the part by inserting mutations in the RBS and RFP. The mutations were found by software, and were inserted into the plasmid by us in the lab. Improvement of RFP expression was measured by fluorescence intensity and compared between this part and our improvement. Measurements also included bacteria with non floursoencence plasmid as negative control and LB as blank.
Results:
Our part improved the expression of mRFP by almost 3 times!
Negative Control Mean- 26,766.72; STDEV- 3423.982
Original Part Mean- 57,853.17; STDEV- 2624.755
Our Part Improvement Mean- 154,336.4; STDEV- 8994.965
Link to the full part improvement notebook:https://2019.igem.org/wiki/images/7/77/T--TAU_Israel--Part_improvment_Notebook.pdf
Link to our part improvement: https://parts.igem.org/Part:BBa_K2943902
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 617
Illegal AgeI site found at 729 - 1000COMPATIBLE WITH RFC[1000]
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