Team:SCUT/Basic Part
Basic Part
Basic Part:BBa_K1724000
In 2015, our team tend to apply for the best basic part. Here's the data of our part.
Introduction
CadA promoter is a cadmium-sensitive promoter. Its repressor is MerR. In high concentration of cadmium, the Cd2+ can rapidly bind with MerR and remove its inhibition of CadA promoter. The reverse is the opposition.
1.Promoter CadA quantification
In 2015 SCUT team, we expressed our three proteins by the downstream of promoter CadA. Promoter CadA is Cd-activated promoter with the presence of MerR. When enough Cadmium existed, the gene downstream can be express. We construct MerR/CadA operon and place the RFP behind the CadA promoter. As the detection limitation mentioned below, the lowest concentration of the cadmium ion is between 10^-8 mol/L and 3*10^-8 mol/L. So we set up the gradient concentration(0, 3*10^-8,10^-7,10^-6,10^-5,10^-4 mol/L) of the cadmium ion in order to get the pattern of our promoter.Our plate reader
Modal: Infinite M200 with the software Magellan 6.5.
Excitation wavelength
485nm
Emission wavelength
528nm
Protocol
Construction
Two required devices J23101 and J23106 were got by overlap PCR. J23117 was cloned from the specified parts in the registry. The three construction were inserted I13504 as a back insert into the promoters. We employed I20270 as positive control, R0040 and TOP 10 without any plasmid as negative control.
Growing
The organism containing the device and three control organisms were streaked out into an agar plates and incubated for 24 hours, which individual colonies are clearly visible. The agar in this step is LB Agar supplemented with 25μg/ml of chloramphenicol. And we incubated liquid culture with our experimental devices and controls, shaking at 250 rpm for 16 hours.
Measuring
After growing, cultures were diluted to an OD600 at 0.5. Measured the fluorescence after setting the plate reader, which the Excitation wavelength was 485nm, the Emission wavelength was 528nm.
Data processing
We measured three times for each biological replicate. The data we reporting was the arithmetic mean of the three data. For the measuring was over for J23101+I13504 in an OD600 of 0.5, we diluted the cultures for four times. So the final data of J23101+I13504 was four times of the raw data.
Calibration curve
Initially, 250mg fluorecein were dissolved into 500ml Phosphate Buffered Saline pH 7.4 (PBS) at a concentration of 500mg/ml, which served as mother liquid. The solution was diluted into varying concentrations (70ng/ml, 60ng/ml, 50ng/ml, 25ng/ml, 10ng/ml, 5ng/ml and PSB only) by PBS. Confirming a linear relationship between fluorescence of sodium fluorescein at different concentrations and calculating the conversion factor as an average across the different concentrations will provide a more accurate control to convert to absolute fluorescence. The plate was read in the plate reader using the settings described previously. Data was transferred to Excel (Microsoft Office 2007) for analysis.
Result
We streaked out the three device and three control organisms into an agar plate and incubated overnight until the individual colonies were clearly visible.
Table 1: This was the time of our samples growing, mostly in 24 hours.
Then devices and controls were incubated liquid culture in 10 ml LB for 16 hours, shaking at 250 rpm, 37 ℃.
Table 2: This was time of growing, mostly in 16 hours. In addition, the culture was interrupted because of power outages. During the blackout, the culture was put on the ice.
After overnight growing, we diluted the liquid culture with LB to OD at 0.5 and measured the florescence three times for every culture.
Here are the data of J23101+I13504. For the measurement of this device in our plate reader was over, the data in this part was four times of the row data, as we diluted the culture for four times.
Figure 1: Measurements after removing background and multiplying for four times for three biological replicate of J23101+I13504. It could be seen that J23101 was a medium high strength of the promoter.
From the result of J23101+I13504, it could be seen that J23101 was a medium high strength of the promoter. And as a result was four times the measured value, the difference between these three data was also relatively large, which resulted in a large standard deviation.
Here are the data of J23106+I13504.
Figure 2: Measurements after removing background for three biological replicate of J23101+I13504. It could be seen that J23106 was a medium low strength of the promoter.
This was the result of J23106+I13504 after removing background control LB. From figure 2 we could see that J23106 was a medium promoter.
Here are the data of J23117+I13504.
Figure 3: Measurements after removing background for three biological replicate of J23117+I13504. It could be seen that J23117was a weak promoter.
This was the measurements of J23117+I13504 after removing the background control LB. It could be seen that J23117 was such a weak promoter that the expression of GFP was low.
Here are the data of positive control I20270.
Figure 4: Measurements after removing background for three biological replicate of our positive control I20270. It showed that there were no false negative results caused by the operation or low content.
Here are the data of negative control R0040
Figure 5: Measurements after removing background for three biological replicate of our negative control R0040. It showed that there were no false positive results caused by the operation or low content.
Here are the data of negative control TOP 10 without any plasmid.
Figure 6: Measurements after removing background for three biological replicate of our negative control TOP 10 without any plasmid. It showed that there were no false positive results caused by the operation or low content and the noise from cells could be nearly ignored.
Figure 7: A linear relationship was confrimed between the concentration of fluorescein(from 5ng/ml to 70 ng/ml) and fluorescence measurement(from 3800 to 51100)
The sodium fluorescein control plate was set up as described previously, and measured with same settings as for characterization samples. A linear relationship was confirmed showing that it was an accurate way representing absolute fluorescence in terms of equivalent ng/ml of sodium fluorescein, at least for the range covered in this calibration curve.
Discussion
Figure 8: standard deviation and fluorescence of the three devices.The standard deviation of J23101+I13504 was relatively larger than the other, for it was four times of the row data and four times of random error as well as machine error. In spite of this, the Relative standard deviation of J23101+I13504 was lower. Besides, the standard deviation race with the increasing of fluorescence.