Revision as of 15:56, 15 October 2019

Medium promoter with medium RBS and GFP

Medium promoter from the constitutive promoter family combined with medium RBS (PR5) and GFP. To quantify the gene expression, GFP was tagged to the promoter RBS domain.

The GFP fluorescence was measured with a plate reader:

BSA calibration line

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.

GFP served as a reporter of expression. We wanted to know how strong the promoter and RBS activity is. With this reporter gene it was possible to analyze the expression via plate reader. GFP is excited at a wavelength of 509nm and has an emission of 520nm. The plate reader illuminates the samples with a high energy xenon flash lamp. Optical filters or monochromator create the exact wavelength. The more GFP in the sample the higher is the GFP fluorescence intensity. The intensity is collected with the second optical system and is detected with a side window photomultiplier tube.

GFP fluorescence intensity dependent on the strenght of promoter and RBS

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)

The results of this test show that PR5 has 28.5 times higher expression of GFP in comparison with with PR3 which has the lowest expression. The fluorescence intensity of GFP varies, and because of lack of time we could not repeat this experiment. We have also tested the promotor and RBS activity with RFP as a reporter and the results deviate from this experiment. So we are looking forward to test this system another time.

SASTRA_Thanjavur 2019 Characterization - Effect of pH on promoter strength

In our experiment, we decided to study the effect of pH on biobrick BBa_K608011. This biobrick consists of a medium constitutive promoter (Bba_J23110) and a medium RBS (Bba_B0032) along with a reporter Green Fluorescent Protein (Bba_E0040).

We began our experiment with the transformation of the biobrick into E. coli DH5alpha cells. We then ventured into identifying the effect of pH on the biobrick’s activity. We know that the expression of the reporter protein relies on the plasmid copy number, binding affinity of ribosomes and RNA polymerase. These mechanisms depend on various environmental conditions including pH. We hypothesized that the expression of GFP would be maximum at an optimal pH of 7.2 and would subsequently decrease as the pH deviated from the optimal one, thus producing a bell-shaped curve when the pH is plotted against the GFP intensity.

This biobrick is present in plasmid pSB1C3, which harbours a chloramphenicol resistant gene for the selection of our transformants. Following the selection of our transformants, we decided to optimize the antibiotic concentration to achieve a balance between cell growth and plasmid selection.

Effect of antibiotic concentration on GFP expression

We found out that the chloramphenicol concentration of 20ug/mL was the optimal concentration for our transformants and decided to keep this as our standard for all further experiments.

To characterize the biobrick, we decided to test its activity under different pH conditions. To achieve the optimal absorbance, our transformants were grown for 48 hours due to their slow growth rate, which can be attributed to the metabolic burden the plasmid imposes on the cells. The fluorescence was measured using a spectrofluorometer with excitation and emission wavelengths of 485nm and 520 nm respectively. This step was done to detect transformants that expressed GFP. Our experiment was performed in triplicates with LB media as our control to measure the background fluorescence.

Effect of pH on Bba_K608011 activity

Our results indicate that the fluorescence intensity was maximum at pH 7.2 (the standard pH of LB broth). Surprisingly, the intensity at pH 8 did not vary significantly from the intensity at pH 7.2 while the intensity at pH 6 deviated much more drastically. At pH values of 5 and 9, we found that the cell growth was extremely low and the absorbance at 595nm was similar to that of LB broth. However, we still proceeded to measure the fluorescence intensity at all 5 pH conditions.
From our experiment, we were able to conclude that pH 7.2 and 8 were optimal conditions for the expression of our biobrick. On the other hand, pH 6 was found to be suboptimal for GFP production. We suspect that this variation in GFP intensity could be due to the effect of pH on the plasmid copy number and the binding affinity of proteins involved in the transcription and translation machineries, such as RNA polymerases and ribosomes.