Difference between revisions of "Part:BBa K608010"

Revision as of 20:12, 18 October 2019

Medium promoter with strong RBS and GFP

Medium promoter from the constitutive promoter family combined with a strong RBS (PR4) and tagged with GFP to quantify the gene expression.

The GFP fluorescence was measured with a plate reader:

Figure 1 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.

Figure 2 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 PR4 has 3.2 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 promoter 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.

Characterization: PR4 vs. T7

Figure 3. Fluorescent colonies of E. coli expressing PR4, indicating successful transformation and production of GFP.

Figure 4. Fluorescein standard curve used to quantify fluorescent intensity. Slight discrepancy from the linear trend at high concentration is most likely due to oversaturation of signal.

Figure 5. Fluorescein log curve used to quantify fluorescent intensity. This was used to quantify fluorescence of GFP signal.

Figure 6. Fluorescent intensity of BL21 expressing GFP under T7 (blue), and PR4 (Green). The T7 promoter lead to about 2.6 as much protein being produced over a 16 hour timescale. Fluorescent intensity was determined using the fluorescein standard and logarithmic curves.

Group: Queens_Canada, 2019
Author: Ruben Warkentin

Summary: We compared the amount of GFP expressed under a constitutive promoter (medium promoter, strong RBS) to T7 expression. Some proteins fold better under constitutive promoters; however, nobody had yet directly compared the amount of protein produced between constitutive vs. T7 expression.

Methods
BioBricks were transformed and expressed in E. coli (BL21). Successful transformation was indicative from the fluorescent E. coli colonies (Fig. 3). BL21 cells were cultured to an OD600=0.6 and 100 uL of culture was transferred into a 96 well plate. Colonies were transfered in quadruplicate. The fluorescence intensity of GFP was measured with a multi-mode microplate reader. The iGEM standardized fluorescence protocol was used for fluorescence measurement standardization (Fig. 4,5; https://www.protocols.io/view/calibration-protocol-plate-reader-fluorescence-cal-6zrhf56).

Results

We found that the T7 promoter produced about 2.6 times as much fluorescent signal as the constitutive PR4 promoter, indicating that T7 is much more efficient at producing GFP (Fig. 6). Interestingly, the production of GFP under PR4 did not increase beyond the level observed at 4 hours after inoculation. It seems that PR4 leads to an initial production of protein; however, after the initial expression the promoter seems to be shut off.

Promoter and RBS:
PR4: medium Promoter (J23110) strong RBS (B0034)
T7: T7 Promoter (BBa_I746909)

Future Directions Future experiments should address the lack of increase in expression over time for the PR4 constitutive promoter. Additionally, other constitutive promoters should be directly compared to T7 promoter expression. All fluorescent data should be collected as per the iGEM standardized fluorescein protocol.

Characterization: Average Fluorescence per Cell

Figure 1.

Figure 2.

Figure 3.

Group: FDR-HB_Peru

Author: Marry Xuan

Summary: We compared parts BBa_K608010 and BBa_K608012 to test if the strength of the RBS correlates to the average amount of fluorescence per cell.

Method:

1. Dilute 10 ul of the culture (BBa_K608010 and BBa_K608012) into 90ul of M9 + Glucose (0.2% w/v) + CAS amino acids (0.2%) medium in the plate reader wells
2. Use a plate reader to read the optical density at 600nm (OD).
3. Use that data to calculate the dilution of the culture to reach an OD reading of 0.1 and dilute with medium (M9 + Glucose + CAS).
4. Take 200 µl of the previous dilution and add it to a well on the plate reader (fill 3 wells for both K608010 and K608012)
5. Fill 3 wells with blank medium.
6. Put the plate in the plate reader and collect the data for 345 minutes, reads both OD and Fluorescence (We used a low gain since the fluorescence was too intense).
7. Once the data was collected, the OD and Fluorescence data was normalized by subtracting the blank. Then to calculate the average fluorescence per cell, we used the following equation: (FLOnorm)/(ODnorm)

Results: We found out that after 345 minutes, the average fluorescence of the GFP with a strong RBS (BBa_K608010) was 2.18 times greater than the average fluorescence of the GFP with a weak RBS (BBa_K608012). This data counters the data from the original experiment performed, where the fluorescence intensity of BBa_K608010 was 5.86 times lower than the fluorescence intensity of BBa_K608012.