Latest revision as of 00:51, 4 September 2020

Medium promoter with weak RBS and GFP

Medium promoter from the constitutive promoter family combined with a weak RBS (PR6) and tagged with GFP to quantify the gene expression.

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 PR6 has 18.6 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.

Tongji_China 2019 Characterization

Our team tested the GFP fluorescence intensity and positive rate of PR3, PR5 and PR6 in E. coli TOP10 with a flow cytometry fluoresce (CytoFLEX LX, Beckman Coulter).
Protocol:
1. The plasmids obtained from 5L,5M,5O,plate 1, are respectively used to transform the TOP10 strain and coated on the LB plate with chloramphenicol. The kanamycin resistant plasmid pET-28a(+) is transformed into the TOP10 strain, which is coated on the LB plate with kanamycin as a negative control. The strain is cultured overnight at 37°C.
2. Pick 3 colonies each plates and culture them in 5 mL LB medium with chloramphenicol 12 hours.
3. 4800 rmp/min centrifugate 10 minutes，using 1.5 mL PBS to dilute the sediment.
4. Using the negative process control sample, adjust forward-scatter and side-scatter voltages to place the strong cell peak as close to the center of the detector range as possible.
5. Instrument gating should be set to ensure that no cell events are discarded.
6. 95% negative control samples’ GFP B525-FITC-A values are less than 104. Regard those samples the GFP B525-FITC-A values more than 104 are positive.
7. Collect at least 5000 events per sample.
Summary: The results of this test show that in E. coli TOP10, PR5 has the highest GFP fluorescence intensity and positive rate among PR2, PR5 and PR6.

Characterization result of BBa_K608008, BBa_K608011 and BBa_K608012, on fluorescence intensity.

Characterization result of BBa_K608008, BBa_K608011 and BBa_K608012, on postive rate.

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.

Sequence and Features

Functional Parameters

Team University of Edinburgh 2019

Group: University of Edinburgh OG, 2019
Author: Francisco Ivan Rodriguez Jaubert and Cathal O’Reilly

Summary: We compared the amount of GFP expressed under the medium constitutive promoter with strong, medium and weak RBS/GFP (PR4-GFP, PR5-GFP, PR6-GFP) in TOP10, BL21 (DE3) and DH5α cells lines. We were interested in replicating the original characterisation as the Freiburg 2011 team mentioned it needed to be replicated. We did so with 3 biological replicates and 3 technical replicates for each control. Additionally, we characterised these biobricks across different common E. coli strains to investigate the consistency across these strains.

Methods
Biobricks were extracted and amplified from the iGEM kit 2019 following standard protocols. Transformation was performed following heat-shock standard protocols. Three positive colonies were picked containing each biobrick in each different cell line. The samples were repeated in series of three to ensure consistency. This was carried out by adding 200 μl of cell culture to wells of a 96-well microplate. The plate reader was set at 485nm wavelength and 520nm emission for analysis.

Results

The next graph shows the average of the three samples (repeated three times) for each biobrick (PR4-GFP, PR5-GFP, and PR6-GFP) in different strains with their respective GFP fluorescence intensity. The weakest RBS (PR6-GFP) in BL21 presents the highest GFP absorbance, in contrast with the Freiburg data showing that the medium RBS (PR5-GFP) had the highest GFP absorbance.

Our data suggests that in BL21 the highest GFP expression was led by the weakest RBS (PR6-GFP), followed by the strongest RBS (PR4-GFP), and finally the medium RBS (PR5-GFP).

TOP10 shows the highest GFP expression with the strongest RBS (PR4-GFP), followed by the medium RBS (PR5-GFP), and finally the weakest RBS (PR6-GFP).

DH5a shows the highest GFP expression with the strongest RBS (PR4-GFP), followed by the weakest RBS (PR6-GFP), and finally the medium RBS (PR5-GFP).

TOP10 is the only cell line showing consistency in the hierarchy of the strongest to the weakest RBS.

Functional Parameters: Austin_UTexas

Burden Imposed by this Part:

Burden is the percent reduction in the growth rate of E. coli cells transformed with a plasmid containing this BioBrick (± values are 95% confidence limits). This part exhibited a significant burden. Users should be aware that BioBricks with a burden of >20-30% may be susceptible to mutating to become less functional or nonfunctional as an evolutionary consequence of this fitness cost. This risk increases as they used for more bacterial cell divisions or in larger cultures. Users should be especially careful when combining multiple burdensome parts, as plasmids with a total burden of >40% are expected to mutate so quickly that they become unclonable. Refer to any one of the BBa_K3174002 - BBa_K3174007 pages for more information on the methods and other conclusions from a large-scale measurement project conducted by the 2019 Austin_UTexas team.

This functional parameter was added by the 2020 Austin_UTexas team.