Team:Hong Kong HKUST/pneumosensor/characterization

From 2014.igem.org

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<p><u><b>Characterization Procedure</b></u><br><br>
 
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1. Constructing :<br>
 
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- σ<sup>x</sup> Generator (BBa_K1379006)-P<sub>celA</sub>-BBa_E0240-pSB3K3 <br>
 
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- σ<sup>x</sup> Generator (BBa_K1379006)-P<sub>comFA</sub>-BBa_E0240-pSB3K3 <br>
 
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- Transforming P<sub>celA</sub>-BBa_E0240-pSB3K3<br>
 
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- Transforming P<sub>comFA</sub>-BBa_E0240-pSB3K3<br>
 
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- Transforming BBa_I20260-pSB3K3 (Standard Constitutive Promoter/Reference Promoter) from the 2014 Distribution Kit<br> - Transforming BBa_E0240-pSB3K3 (GFP generator) from the 2014 Distribution Kit.<br><br>
 
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2. Preparing supplemented M9 medium (M9 Minimal salt medium protocols could be seen on the protocols page) <br><br>
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<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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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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3. Transforming P<sub>celA</sub>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>-pSB3K3<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>
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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> 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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3. Culturing <i>E. coli</i> DH10B strain carrying the whole construct listed on procedure number 1, in supplemented M9 medium and measuring the respective growth curve; <br><br>
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<p><u><b>Measurement</b></u><br><br>
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1. Preparing supplemented M9 medium (M9 Minimal salt medium protocols could be seen on the protocols page) <br><br>
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4. Measuring the GFP intensity and OD595 values every 30 minutes after the above mentioned E. coli strains are cultured to mid-log phase; <br><br>
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2. Culturing <i>E. coli</i> DH10B strain carrying the whole construct listed on procedure number 1, in supplemented M9 medium and measuring the respective growth curve; <br><br>
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5. Calculating the Relative Promoter Units (RPU) using the obtained data; <br><br>
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3. Measuring the GFP intensity and OD595 values every 30 minutes after the above mentioned E. coli strains are cultured to mid-log phase; <br><br>
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4. Calculating the Relative Promoter Units (RPU) using the obtained data; <br><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 BBa_E0240-pSB3K3. Curve reflecting GFP expression change was plotted; OD595 was converted to OD600, and average values were taken; <br><br>
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2. GFP intensity are subtracted with the background fluorescence which is <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>-pSB3K3. Curve reflecting GFP expression change was plotted; OD595 was converted to OD600, and average values were taken; <br><br>
3. GFP synthesis rate was then obtained by calculating the slope of the above mentioned curve; <br><br>
3. GFP synthesis rate was then obtained by calculating the slope of the above mentioned curve; <br><br>
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4. Absolute promoter activity of P<sub>celA</sub>, P<sub>comFA</sub>, and BBa_I20260 were calculated by dividing the GFP synthesis rate with the average OD600 value; <br><br>
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4. Absolute promoter activity of P<sub>celA</sub>, P<sub>comFA</sub>, and <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> were calculated by dividing the GFP synthesis rate with the average OD600 value; <br><br>
5. Averaged absolute promoter activity was then obtained by averaging the respective 3 sets of absolute promoter activity values; <br><br>
5. Averaged absolute promoter activity was then obtained by averaging the respective 3 sets of absolute promoter activity values; <br><br>
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6. Finally, R.P.U was calculated by dividing the averaged P<sub>celA</sub> and P<sub>comFA</sub> absolute promoter activity over the averaged BBa_J23101 absolute promoter activity. R.P.U value of P<sub>celA</sub> and P<sub>comFA</sub> reflect the maximum GFP expression in the presence of σ<sup>x</sup>. Leakage could be analyzed according to the R.P.U value that shows the GFP expression of P<sub>celA</sub> and P<sub>comFA</sub> promoter in the absence of σ<sup>x</sup>.<br><br>
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6. Finally, R.P.U was calculated by dividing the averaged P<sub>celA</sub> and P<sub>comFA</sub> absolute promoter activity over the averaged <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> absolute promoter activity. R.P.U value of P<sub>celA</sub> and P<sub>comFA</sub> reflect the maximum GFP expression in the presence of σ<sup>x</sup>. Leakage could be analyzed according to the R.P.U value that shows the GFP expression of P<sub>celA</sub> and P<sub>comFA</sub> promoter in the absence of σ<sup>x</sup>.<br><br>
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Revision as of 14:12, 13 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

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 (BBa_K1379006)-PcelA-BBa_E0240-pSB3K3
2. Construct σx Generator (BBa_K1379006)-PcomFA-BBa_E0240-pSB3K3
3. Transforming PcelA-BBa_E0240-pSB3K3
4. Transforming PcomFA-BBa_E0240-pSB3K3
5. Transforming BBa_I20260-pSB3K3 (Standard Constitutive Promoter/Reference Promoter) from the 2014 Distribution Kit
6. Transforming BBa_E0240-pSB3K3 (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)

2. Culturing E. coli DH10B strain carrying the whole construct listed on procedure number 1, in supplemented M9 medium and measuring the respective growth curve;

3. Measuring the GFP intensity and OD595 values every 30 minutes after the above mentioned E. coli strains are cultured to mid-log phase;

4. 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 BBa_E0240-pSB3K3. Curve reflecting GFP expression change was plotted; 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.


References

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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