Difference between revisions of "Team:SCUT/Basic Part"

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<h3>1.Promoter CadA quantification</h3>
 
<h3>1.Promoter CadA quantification</h3>
 
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.<br/>
 
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.<br/>
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Our plate reader<br/>
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<p id="img"><https://static.igem.org/mediawiki/parts/f/f4/2015result_1.jpg"></p>
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Modal: Infinite M200 with the software Magellan 6.5. <br/>
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Excitation wavelength<br/>
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485nm<br/>
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Emission wavelength<br/>
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528nm<br/>
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<h3>Protocol</h3>
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<p>Construction<br/>
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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. <br/>
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Growing<br/>
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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. <br/>
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Measuring<br/>
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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.  <br/>
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Data processing <br/>
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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.<br/>
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Calibration curve<br/>
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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.<br/>
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</p>
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<p>
 
<p>
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<h3>Result</h3>
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Figure 1:  From left and right the concentration of cadmium chloride which is added into medium is 0,3*10^-8,10^-7,10^-6,10^-5,10^-4 mol/L. We can see that the medium become red with the presence of cadmium ion in contrast of the absence of the cadmium. This is the visible test how different concentration of cadmium ion activates the CadA/MerR operon.<br/>
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We streaked out the three device and three control organisms into an agar plate and incubated overnight until the individual colonies were clearly visible.<br/>
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</p>
 
</p>
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<p id="img"><img src="https://static.igem.org/mediawiki/parts/7/72/2015SCUT-Mtable-1.png"></p>
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<p id="img"><img src="https://static.igem.org/mediawiki/parts/e/e3/2015result_2.jpg"></p>
 
<p>
 
<p>
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Table 1: This was the time of our samples growing, mostly in 24 hours.<br/>
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Figure 2. Accumulation of RFP fluorescence in culture of engineering E.coli.The promoter CadA Strength test in the concentration of 10^-6 mol/L and 0mol/L cadmium chloride and 0mol/L. We construct Constructive promoter +MerR+termintor & PCadA+CsgA-EC(n)+RFP+tetR+termintor into E.coli. The abscissa is the time after we add the cadmium chloride . The ordinate is RLU/OD. The curve indicates that the engineering E.coli can be activated and go into stabilization phase at 9 hours.<br/>
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Then devices and controls were incubated liquid culture in 10 ml LB for 16 hours, shaking at 250 rpm, 37 ℃.<br/>
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</p>
 
</p>
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<p id="img"><img src="https://static.igem.org/mediawiki/parts/8/86/2015SCUT-Mtable-2.png"></p>
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<p id="img"><img src="https://static.igem.org/mediawiki/parts/9/96/2015result-2.png"></p>
 
<p>
 
<p>
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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.<br/>
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Figure 2-2. The brief introduction of plasmid of the CadA promoter quantification.<br/>
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After overnight growing, we diluted the liquid culture with LB to OD at 0.5 and measured the florescence three times for every culture.<br/>
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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. <br/>
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</p>
 
</p>
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<p id="img"><img src="https://static.igem.org/mediawiki/parts/5/5f/2015SCUT-measurement-1.png" alt=" " /></p>
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<h3>2.Detection limit of promoter CadA</h3>
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<p>
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In order to test the detection limit of the promoter CadA, we first set up gradient concentration of Cadmium（10^-8,10^-7,10^-6,10^-5,10^-4mol/L）to estimate the probably range. As the result mentioned above, the promoter CadA can be activated over the concentration of 3*10^-8mol/L. So we set up the gradient of Cd2+ (3*10^-9, 10^-8, 3*10^-8, 5*10^-8mol/L) and obtain the detection limit of promoter CadA.
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<p id="img"><img src="https://static.igem.org/mediawiki/parts/7/7c/2015result_3.JPG" alt=" " /></p>
 
<p>
 
<p>
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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.<br/>
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Figure 3. The visible test of detection limitation. The flag on the 50ml tube was the concentration of cadmium ion. As seen in the picture, 3*10^-9 mol/L and 1*10^-8 mol/L Cd2+ cadmium medium didn't change into red contrast of 3*10^-8 mol/L and 5*10^-8mol/L. The minimum motivating concentration is between 10^-8mol/L and 3*10^-8 mol/L.<br/>
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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.<br/>
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Here are the data of J23106+I13504.<br/>
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</p>
 
</p>
  
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<p id="img"><img src="https://static.igem.org/mediawiki/parts/2/23/2015SCUT-measurement-2.png" alt=" " /></p>
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<p id="img"><img src="https://static.igem.org/mediawiki/parts/d/d5/2015result_4.png" alt=" " /></p>
 
<p>
 
<p>
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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.<br/>
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Figure 4. The actual data of activation limit of promoter CadA after inducing within 21 hours. Lower than 10^-8 mol/L, the cadmium cannot motivate the operon CadA/MerR, contrast of the higher concentration. Meanwhile, more than 10^-7 mol/L, the expression of RFP stays steady, the maximum is near 11000 RLU/OD.
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This was the result of J23106+I13504 after removing background control LB. From figure 2 we could see that J23106 was a medium promoter.
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So we can come to a conclusion that the lowest concentration of cadmium to activate promoter CadA is between 10^-8 and 3*10^-8 mol/L.<br/>
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Here are the data of J23117+I13504.<br/>
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</p>
 
</p>
 
<p id="img"><img src="https://static.igem.org/mediawiki/parts/6/63/2015SCUT-measurement-3.png" alt=" " /></p>
 
<p id="img"><img src="https://static.igem.org/mediawiki/parts/6/63/2015SCUT-measurement-3.png" alt=" " /></p>
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Here are the data of negative control R0040<br/>
 
Here are the data of negative control R0040<br/>
 
</p>
 
</p>
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<p id="img"><img src="https://static.igem.org/mediawiki/parts/6/6d/2015SCUT-measurement-5.png" alt=" " /></p>
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<p>
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<h3>3. The specificity of the CadA promoter</h3>
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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.<br/>
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Here are the data of negative control TOP 10 without any plasmid.<br/>
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</p>
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<p id="img"><img alt=" " src="https://static.igem.org/mediawiki/parts/2/2b/2015SCUT-measurement-6.png" /></p>
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<p>
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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.<br/>
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</p>
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<p id="img"><img alt=" " src="https://static.igem.org/mediawiki/parts/5/5b/2015SCUT-measurement-7.png" /></p>
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<p>
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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)
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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. <br/>
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</p>
 
</p>
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<p id="img"><img src="https://static.igem.org/mediawiki/parts/9/9f/2015result-specific.png" alt=" " /></p>
 
<p>
 
<p>
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<h3>Discussion</h3>
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Figure 5. The specificity of the MerR/CadA promoter. As seen in the picture, the medium added cadmium in became red, in contrast that the medium which was added nothing or added other heavy metal maintained the same situation.<br/>
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Figure 8: standard deviation and fluorescence of the three devices. <br/>
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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.<br/>
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</p>
 
</p>
 
</div>
 
</div>

Revision as of 19:46, 18 September 2015

Team:SCUT

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.

Figure 1: From left and right the concentration of cadmium chloride which is added into medium is 0,3*10^-8,10^-7,10^-6,10^-5,10^-4 mol/L. We can see that the medium become red with the presence of cadmium ion in contrast of the absence of the cadmium. This is the visible test how different concentration of cadmium ion activates the CadA/MerR operon.

Figure 2. Accumulation of RFP fluorescence in culture of engineering E.coli.The promoter CadA Strength test in the concentration of 10^-6 mol/L and 0mol/L cadmium chloride and 0mol/L. We construct Constructive promoter +MerR+termintor & PCadA+CsgA-EC(n)+RFP+tetR+termintor into E.coli. The abscissa is the time after we add the cadmium chloride . The ordinate is RLU/OD. The curve indicates that the engineering E.coli can be activated and go into stabilization phase at 9 hours.

Figure 2-2. The brief introduction of plasmid of the CadA promoter quantification.

2.Detection limit of promoter CadA

In order to test the detection limit of the promoter CadA, we first set up gradient concentration of Cadmium（10^-8,10^-7,10^-6,10^-5,10^-4mol/L）to estimate the probably range. As the result mentioned above, the promoter CadA can be activated over the concentration of 3*10^-8mol/L. So we set up the gradient of Cd2+ (3*10^-9, 10^-8, 3*10^-8, 5*10^-8mol/L) and obtain the detection limit of promoter CadA.

Figure 3. The visible test of detection limitation. The flag on the 50ml tube was the concentration of cadmium ion. As seen in the picture, 3*10^-9 mol/L and 1*10^-8 mol/L Cd2+ cadmium medium didn't change into red contrast of 3*10^-8 mol/L and 5*10^-8mol/L. The minimum motivating concentration is between 10^-8mol/L and 3*10^-8 mol/L.

Figure 4. The actual data of activation limit of promoter CadA after inducing within 21 hours. Lower than 10^-8 mol/L, the cadmium cannot motivate the operon CadA/MerR, contrast of the higher concentration. Meanwhile, more than 10^-7 mol/L, the expression of RFP stays steady, the maximum is near 11000 RLU/OD. So we can come to a conclusion that the lowest concentration of cadmium to activate promoter CadA is between 10^-8 and 3*10^-8 mol/L.

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

3. The specificity of the CadA promoter

Figure 5. The specificity of the MerR/CadA promoter. As seen in the picture, the medium added cadmium in became red, in contrast that the medium which was added nothing or added other heavy metal maintained the same situation.

About Us

In 2015, we SCUT teams won top ten innovative and entrepreneurial team set up by SCUT.Because of the strong support of the college, our team is being on the right track, and increasing understanding of the subject and experience.

Thanks

  • Zhang Zhenwu,Prof. Guo Shouqian,Dr. Li, Dr. Li Cheng,Dr. Wang Meng,Chen Kejie
  • Guangzhou Municipal Environmental Protection Bureau

COPYRIGHT ©2015-SCUT