Team:Hong Kong HKUST/pneumosensor/characterization

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<p class="first_letter_enhanced">To measure the R.P.U (Relative Promoter Unit) of P<sub>celA</sub> promoter, we adopted the method described by Kelly et al. in “Measuring the activity of BioBrick promoters using an in vivo reference standard” (Kelly et al., 2009). By linking P<sub>celA</sub> promoter with GFP generator (BBa_E0240), the promoter activity was represented by the GFP synthesis rate. Fluorescence was measured using EnVision multilabel reader from Perkin Elmer Company, when the cells are in the mid-log phase. OD595 values were measured and converted to OD600 to obtain the R.P.U of the promoter. The σ<sup>x</sup> gene and P<sub>celA</sub> promoter used in the construct are both cloned from <i>Streptococcus pneumoniae</i> NCTC 7465 strain. The experimental construct used was BBa_K1379005, which contain σ<sup>x</sup> generator, P<sub>celA</sub>, and GFP generator. The positive control used in this characterization is BBa_I20260 which is a constitutive promoter containing GFP generator, while the negative control used in this characterization is BBa_K1379002 which is P<sub>celA</sub> promoter with GFP generator but without σ<sup>x</sup> generator.<br> </p>
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<p class="first_letter_enhanced"> For characterization, PcelA promoter was assembled with the promoter measurement kit <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> to give the PcelA Measurement Kit <a href= "http://parts.igem.org/Part:BBa_K1379002">BBa_K1379002</a> in plasmid pSB3K3. The construct was further assembled with &sigma;<sup>X</sup> generator <a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a> to give <a href= "http://parts.igem.org/Part:BBa_K1379005">BBa_K1379005</a>.
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Qualitative characterization was performed by comparing intensities of GFP signals from colonies of <i>E. coli</i> DH10B strain holding the P<sub>celA</sub> Measurement Kits with and without the &sigma;<sup>X</sup> generator under a fluorescent macroscope with UV filter. Measurement kit for standard reference promoter <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>, <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> was used as a positive control; <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> was used as the negative control for background fluorescence. <br><br>
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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). <i>E. coli</i> DH10B strains holding the constructs with or without &sigma;<sup>X</sup> 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 <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> measurement device <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> to obtain the Relative Promoter Units (RPUs). For subtraction of background fluorescence, pSB3K3 holding <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> was measured alongside. The measurement was done with 3 replicas.
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<img src= "http://2014.igem.org/wiki/images/3/35/PcelA_wiki_large.png"/><br>
<img src= "http://2014.igem.org/wiki/images/3/35/PcelA_wiki_large.png"/><br>
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<h5 style="font-size: 13px">Figure 2. P<sub>celA</sub> promoter Relative Promoter Unit (RPU) is measured with reference to J23101 constitutive promoter.</h5>
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<h5 style="font-size: 13px">Figure 2. P<sub>celA</sub> has 0.53 RPU when paired with &sigma;<sup>X</sup> generator.</h5>
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<h6 style= "font-size: 13px"> P<sub>celA</sub> promoter induced by σ<sup>x</sup> gave GFP signals. This fluorescence expression was measured over time, and divided by the OD of the cells. In the end, it was standardized based on J23101 promoter strength. Measurement was done by using 3 replicas.</h6>
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<h6 style= "font-size: 13px"> P<sub>celA</sub> was measured in reference to <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> constitutive promoter with and without &sigma;<sup>X</sup> generator <a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a>. RPU shown was calculated from 3 replicas.</h6>
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<img src= "http://2014.igem.org/wiki/images/f/fd/PcomFA_prism_large.png"/>
<img src= "http://2014.igem.org/wiki/images/f/fd/PcomFA_prism_large.png"/>
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<h5 style="font-size: 13px">Figure 3. P<sub>comFA</sub> promoter Relative Promoter Unit (RPU) is measured with reference to J23101 constitutive promoter. </h5>
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<h5 style="font-size: 13px">Figure 3. P<sub>comFA</sub> has 1.21 RPU when paired with &sigma;<sup>X</sup> generator. </h5>
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<h6 style= "font-size: 13px"> P<sub>comFA</sub> promoter induced by σ<sup>x</sup> gave GFP signals. This fluorescence expression was measured over time, and divided by the OD of the cells. In the end, it was standardized based on J23101 promoter strength. Measurement was done by using 3 replicas.</h6>
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<h6 style= "font-size: 13px"> P<sub>comFA</sub> was measured in reference to <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> constitutive promoter with and without &sigma;<sup>X</sup> generator <a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a>. RPU shown was calculated from 3 replicas.</h6>
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<p class="first_letter_enhanced">The method of measuring RPU (Relative Promoter Unit) of P<sub>comFA</sub> promoter is similar to measuring RPU of P<sub>celA</sub> which adopted the method described by Kelly et al. in “Measuring the activity of BioBrick promoters using an in vivo reference standard” (Kelly et al., 2009). There are no difference in measurement condition of measuring P<sub>comFA</sub> and P<sub>celA</sub>. Fluorescence and absorbance are measured in the same time period. The experimental construct used was BBa_K1379007, which contain σ<sup>x</sup> generator, P<sub>comFA</sub>, and GFP generator. The positive control used in this characterization is BBa_I20260 which is a constitutive promoter containing GFP generator, while the negative control used in this characterization is BBa_K1379003 which is P<sub>comFA</sub> promoter with GFP generator but without σ<sup>x</sup> generator. </p>
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<p class="first_letter_enhanced">The method of measuring RPU (Relative Promoter Unit) of P<sub>comFA</sub> promoter is similar to measuring RPU of P<sub>celA</sub>. PcomFA promoter was assembled with the promoter measurement kit <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> to give the PomFA Measurement Kit <a href= "http://parts.igem.org/Part:BBa_K1379003">BBa_K1379003</a> in plasmid pSB3K3. The construct was further assembled with &sigma;<sup>X</sup> generator <a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a> to give <a href= "http://parts.igem.org/Part:BBa_K1379007">BBa_K1379007</a>.
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Qualitative characterization was performed by comparing intensities of GFP signals from colonies of <i>E. coli</i> DH10B strain holding the P<sub>comFA</sub> Measurement Kits with and without the &sigma;<sup>X</sup> generator under a fluorescent macroscope with UV filter. Measurement kit for standard reference promoter <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>, <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> was used as a positive control; <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> was used as the negative control for background fluorescence. <br><br>
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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). <i>E. coli</i> DH10B strains holding the constructs with or without &sigma;<sup>X</sup> 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 <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> measurement device <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> to obtain the Relative Promoter Units (RPUs). For subtraction of background fluorescence, pSB3K3 holding <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> was measured alongside. The measurement was done with 3 replicas. </p>
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Revision as of 05:19, 12 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)

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. Qualitative characterization was performed by comparing intensities of GFP signals from colonies of E. coli DH10B strain holding the PcelA 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 when paired with σX generator.
PcelA was measured in reference to BBa_J23101 constitutive promoter with and without σX generator BBa_K1379006. RPU shown was calculated from 3 replicas.

PcomFA(BBa_K1379001)

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

The method of measuring RPU (Relative Promoter Unit) of PcomFA promoter is similar to measuring RPU of PcelA. PcomFA promoter was assembled with the promoter measurement kit BBa_E0240 to give the PomFA 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 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.

Characterization Method

Characterization Procedure

1. Constructing :
- σx Generator (BBa_K1379006)-PcelA-BBa_E0240-pSB3K3
- σx Generator (BBa_K1379006)-PcomFA-BBa_E0240-pSB3K3
- Transforming PcelA-BBa_E0240-pSB3K3
- Transforming PcomFA-BBa_E0240-pSB3K3
- Transforming BBa_I20260-pSB3K3 (Standard Constitutive Promoter/Reference Promoter) from the 2014 Distribution Kit
- Transforming BBa_E0240-pSB3K3 (GFP generator) from the 2014 Distribution Kit.

2. Preparing supplemented M9 medium (M9 Minimal salt medium protocols could be seen on the protocols page)

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

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

5. 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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