Team:Hong Kong HKUST/pneumosensor/characterization

From 2014.igem.org

(Difference between revisions)
Line 92: Line 92:
<p><u><b>Construction</b></u><br><br>
<p><u><b>Construction</b></u><br><br>
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1. Construct σ<sup>x</sup> Generator (<a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a>)-P<sub>celA</sub>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>-pSB3K3<br>
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1. Construct σ<sup>x</sup> Generator pSB3K3-(<a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a>)-P<sub>celA</sub>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br>
Or migrate <a href= "http://parts.igem.org/Part:BBa_K1379005">BBa_K1379005</a> to pSB3k3.<br><br>
Or migrate <a href= "http://parts.igem.org/Part:BBa_K1379005">BBa_K1379005</a> to pSB3k3.<br><br>
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2. Construct σ<sup>x</sup> Generator (<a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a>)-P<sub>comFA</sub>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>-pSB3K3 <br>
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2. Construct σ<sup>x</sup> Generator pSB3K3-(<a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a>)-P<sub>comFA</sub>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> <br>
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Or migrate <a href= "http://parts.igem.org/Part:BBa_K1379007">BBa_K1379007</a> to pSB3k3.<br><br>
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Or migrate <a href= "http://parts.igem.org/Part:BBa_K1379007">BBa_K1379007</a> to pSB3K3.<br><br>
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3. Transforming P<sub>celA</sub>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>-pSB3K3<br><br>
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3. Transforming pSB3K3-P<sub>celA</sub>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br><br>
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4. Transforming P<sub>comFA</sub>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>-pSB3K3<br><br>
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4. Transforming pSB3K3-P<sub>comFA</sub>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br><br>
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5. Transforming <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a>-pSB3K3 (Standard Constitutive Promoter/Reference Promoter) from the 2014 Distribution Kit<br><br>
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5. Transforming pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> (Standard Constitutive Promoter/Reference Promoter) from the 2014 Distribution Kit<br><br>
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6. Transforming <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>-pSB3K3 (GFP generator) from the 2014 Distribution Kit.<br><br>
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6. Transforming pSB3K3<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> (GFP generator) from the 2014 Distribution Kit.<br><br>
<br>
<br>
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1. Preparing supplemented M9 medium <br>(M9 Minimal salt medium protocols could be seen on the <a href= "https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab/protocols">Protocols</a> page, or download the <a href= "https://static.igem.org/mediawiki/2014/8/8b/M9_Minimal_medium_protocol.pdf">PDF</a> file) <br><br>
1. Preparing supplemented M9 medium <br>(M9 Minimal salt medium protocols could be seen on the <a href= "https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab/protocols">Protocols</a> page, or download the <a href= "https://static.igem.org/mediawiki/2014/8/8b/M9_Minimal_medium_protocol.pdf">PDF</a> file) <br><br>
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2. Culturing <i>E. coli</i> DH10B strain carrying the whole construct listed on procedure number 1. Grow cell culture overnight (Incubate 37°C and shake for 15 hours) in falcon tubes with M9 minimal medium; <br><br>
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2. Culturing <i>E. coli</i> DH10B strain carrying the whole construct listed on procedure number 1. Grow cell culture overnight (Incubate 37°C and shake for 15 hours) with M9 minimal medium (we used Corning® 96 well storage system storage block, 2 mL, V-bottom, sterile to culture the cells, and Corning® microplate sealing tape white Rayon (with acrylic), sterile, suitable for cell/tissue culture applications, breathable sterile membrane.)<br><br>
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3. Take out 20-30μl of overnight cell culture with Multichannel Pipetman and mix it with M9 medium in the 96 Deep Well plate. <br><br>
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3. Take out 20-30μl of overnight cell culture (we used Multichannel Pipetman) and mix it with M9 medium in the 96 Deep Well plate. <br><br>
4. Incubate in 37°C and shake for 3 - 4 hours.<br><br>
4. Incubate in 37°C and shake for 3 - 4 hours.<br><br>
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5. Take out 200ul of cells from the 96 deep well plates, and put it on a micro test plate 96 well flat bottom.<br><br>
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5. Take out 200ul of cells from the 96 deep well plates, and put it on a micro test plate 96 well flat bottom. (we used Micro test plate 96 well flat bottom, made by SARSTEDT.)<br><br>
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6. Measuring the GFP intensity and OD595 values every 30 minutes after the above mentioned E. coli strains are cultured to mid-log phase (OD600 = 0.3 - 0.5);<br><br>
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6. Measuring the GFP intensity and OD595 values every 30 minutes after the above mentioned <i>E. coli</i> strains are cultured to mid-log phase (OD600 = 0.3 - 0.5) (we used Envision Multilabel Reader.)<br><br>
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Filter used: <br>
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Filter used on Envision Multilabel Reader: <br>
- Absorbance :Photometric 595nm,<br>
- Absorbance :Photometric 595nm,<br>
- Excitation :485nm FITC,<br>  
- Excitation :485nm FITC,<br>  
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1. After <i>E. coli</i> carrying the right construct was grown to mid-log phase, GFP intensity and OD595 were measured every 30 minutes (up to 120min); <br><br>
1. After <i>E. coli</i> carrying the right construct was grown to mid-log phase, GFP intensity and OD595 were measured every 30 minutes (up to 120min); <br><br>
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2. GFP intensity are subtracted with the background fluorescence which is the fluorescence of <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>-pSB3K3. Curve reflecting GFP expression change was plotted (from 4 measurements from time=0 to time=120); OD595 was converted to OD600, and average values were taken; <br><br>
+
2. GFP intensity are subtracted with the background fluorescence which is the fluorescence of pSB3K3-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>. Curve reflecting GFP expression change was plotted (from 4 measurements from time=0 to time=120); OD595 was converted to OD600, and average values were taken; <br><br>
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<p><br><u><b>Equipments</b></u><br><br>
<p><br><u><b>Equipments</b></u><br><br>
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1. Multichannel Pipetman.<br>
 
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2. Corning® 96 well storage system storage block, 2 mL, V-bottom, sterile. <br>
 
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3. Corning® microplate sealing tape white Rayon (with acrylic), sterile, suitable for cell/tissue culture applications, breathable sterile membrane. <br>
 
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4. Micro test plate 96 well flat bottom, made by SARSTEDT. <br>
 
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5. Envision Multilabel Reader.
 

Revision as of 16:31, 17 October 2014



Pneumosensor Characterization

σx(BBa_K1379004)


Introduction

To test the functionality of σX, we first enable constitutive expression of σX in the σX Generator, BBa_K1379006.The generator was then assembled with the standard promoter measurement kit BBa_E0240, with either promoter PcelA (Promoter only: BBa_K1379000, w/ BBa_E0240: BBa_K1379002) and PcomFA (Promoter only: BBa_K1379001, w/ BBa_E0240: BBa_K1379003). E. coli colonies holding the resulting constructs in pSB3K3 were observed under fluorescent macroscope with UV filter. Measurement kit for standard reference promoter BBa_J23101, which is BBa_I20260 was used as a positive control; BBa_E0240 was used as the general negative control. for background fluorescence. Measurement kits for PcelA PcomFA without σX Generator were used as negative controls for function of σX.


Results

Figure 1. PcelA and PcomFA promoters activated in presence of σX.
Only in the presence of σX would PcelA and PcomFA be turned on, as GFP expression could be seen when σX present. Therefore, σX is functional. PcelA and PcomFA gave little GFP signal in the absence of σX but has comparable activity as reference promoter BBa_J23101 in presence of σX. Scale bar = 5mm.

PcelA (BBa_K1379000) and PcomFA (BBa_K1379001)



For characterization, PcelA promoter was assembled with the promoter measurement kit BBa_E0240 to give the PcelA Measurement Kit BBa_K1379002 in plasmid pSB3K3. The construct was further assembled with σX generator BBa_K1379006 to give BBa_K1379005.

PcomFA promoter was assembled with the promoter measurement kit BBa_E0240 to give the PcomFA Measurement Kit BBa_K1379003 in plasmid pSB3K3. The construct was further assembled with σX generator BBa_K1379006 to give BBa_K1379007.

Qualitative characterization was performed by comparing intensities of GFP signals from colonies of E. coli DH10B strain holding the PcelA and PcomFA Measurement Kits with and without the σX generator under a fluorescent macroscope with UV filter. Measurement kit for standard reference promoter BBa_J23101, BBa_I20260 was used as a positive control; BBa_E0240 was used as the negative control for background fluorescence.

Quantitative characterization was performed following the protocol described in “Measuring the activity of BioBrick promoters using an in vivo reference standard” (Kelly et al., 2009). E. coli DH10B strains holding the constructs with or without σX generator respectively were grown to mid-log phases. GFP intensities and cell densities were then sampled every 30 minutes for 5 consecutive time points to obtain growth rates and GFP synthesis rates. The GFP synthesis rates were then compared to that of standard reference promoter BBa_J23101 measurement device BBa_I20260 to obtain the Relative Promoter Units (RPUs). For subtraction of background fluorescence, pSB3K3 holding BBa_E0240 was measured alongside. The measurement was done with 3 replicas.




Figure 2. PcelA has 0.53 RPU and PcomFA hsa 1.21 RPU when paired with σX generator.
PcelA and PcomFA was measured in reference to BBa_J23101 constitutive promoter with and without σX generator BBa_K1379006. RPU shown was calculated from 3 replicas.

Characterization Method

Construction

1. Construct σx Generator pSB3K3-(BBa_K1379006)-PcelA-BBa_E0240
Or migrate BBa_K1379005 to pSB3k3.

2. Construct σx Generator pSB3K3-(BBa_K1379006)-PcomFA-BBa_E0240
Or migrate BBa_K1379007 to pSB3K3.

3. Transforming pSB3K3-PcelA-BBa_E0240

4. Transforming pSB3K3-PcomFA-BBa_E0240

5. Transforming pSB3K3-BBa_I20260 (Standard Constitutive Promoter/Reference Promoter) from the 2014 Distribution Kit

6. Transforming pSB3K3BBa_E0240 (GFP generator) from the 2014 Distribution Kit.


Measurement

1. Preparing supplemented M9 medium
(M9 Minimal salt medium protocols could be seen on the Protocols page, or download the PDF file)

2. Culturing E. coli DH10B strain carrying the whole construct listed on procedure number 1. Grow cell culture overnight (Incubate 37°C and shake for 15 hours) with M9 minimal medium (we used Corning® 96 well storage system storage block, 2 mL, V-bottom, sterile to culture the cells, and Corning® microplate sealing tape white Rayon (with acrylic), sterile, suitable for cell/tissue culture applications, breathable sterile membrane.)

3. Take out 20-30μl of overnight cell culture (we used Multichannel Pipetman) and mix it with M9 medium in the 96 Deep Well plate.

4. Incubate in 37°C and shake for 3 - 4 hours.

5. Take out 200ul of cells from the 96 deep well plates, and put it on a micro test plate 96 well flat bottom. (we used Micro test plate 96 well flat bottom, made by SARSTEDT.)

6. Measuring the GFP intensity and OD595 values every 30 minutes after the above mentioned E. coli strains are cultured to mid-log phase (OD600 = 0.3 - 0.5) (we used Envision Multilabel Reader.)

Filter used on Envision Multilabel Reader:
- Absorbance :Photometric 595nm,
- Excitation :485nm FITC,
- Emission :535nm FITC,
- Mirror module : FITC (403) at bottom.

- In between measurements, keep incubating the cells in 37°C while shaking.

7. Calculating the Relative Promoter Units (RPU) using the obtained data;


Data Processing

1. After E. coli carrying the right construct was grown to mid-log phase, GFP intensity and OD595 were measured every 30 minutes (up to 120min);

2. GFP intensity are subtracted with the background fluorescence which is the fluorescence of pSB3K3-BBa_E0240. Curve reflecting GFP expression change was plotted (from 4 measurements from time=0 to time=120); OD595 was converted to OD600, and average values were taken;

3. GFP synthesis rate was then obtained by calculating the slope of the above mentioned curve;

4. Absolute promoter activity of PcelA, PcomFA, and BBa_I20260 were calculated by dividing the GFP synthesis rate with the average OD600 value;

5. Averaged absolute promoter activity was then obtained by averaging the respective 3 sets of absolute promoter activity values;

6. Finally, R.P.U was calculated by dividing the averaged PcelA and PcomFA absolute promoter activity over the averaged BBa_J23101 absolute promoter activity. R.P.U value of PcelA and PcomFA reflect the maximum GFP expression in the presence of σx. Leakage could be analyzed according to the R.P.U value that shows the GFP expression of PcelA and PcomFA promoter in the absence of σx.

Equation of the RPU calculation is shown below:



Equipments


References

BioCyc was retrieved from http://www.biocyc.org/SPNE171101/NEW-IMAGE?type=GENE&object=GJC8-867 and http://www.biocyc.org/SPNE171101/NEW-IMAGE?type=GENE&object=GJC8-867

Luo P., & Morrison D. (2003). Transient Association of an Alternative Sigma Factor, ComX, with RNA Polymerase during the Period of Competence for Genetic Transformation in Streptococcus pneumoniae. Journal of Bacteriology. doi:10.1128/JB.185.1.349-358.2003

Piotrowski A., Luo P., & Morrison D. (2009). Competence for genetic transformation in Streptococcus pneumoniae: termination of activity of the alternative sigma factor ComX is independent of proteolysis of ComX and ComW. Journal of Bacteriology. doi:10.1128/JB.01750-08

Rhodius V., Segall-Shapiro T., Sharon B., Ghodasara A., Orlova E., Tabakh H., . . . Voigt C. (2013). Design of orthogonal genetic switches based on a crosstalk map of σs, anti-σs, and promoters. Molecular Systhetic Biology .doi:10.1038/msb.2013.58

J. R. Kelly, A. J. Rubin, J. H. Davis, J. Cumbers, M. J. Czar, ..., D. Endy. (2009). Measuring the activity of BioBrick promoters using an in vivo reference standard. Journal of Biological Engineering, 3, 4. doi: 10.1186/1754-1611-3-4

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