Team:Hong Kong HKUST/pneumosensor/characterization

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<h2>Pneumosensor Characterization</h2>
<h2>Pneumosensor Characterization</h2>
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<div class='content_1'><h3> <a href= "http://parts.igem.org/Part:BBa_K1379004"> σ<sup>x</sup>(BBa_K1379004) </a></h3>
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<h3> <a href= "http://parts.igem.org/Part:BBa_K1379004"> Sigma X (BBa_K1379004) </a></h3>
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<p> <b>Introduction</b> <br><br>
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<p><br><b><u>Introduction</u></b> <br><br>
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In our characterization, <i>SigmaX</i> and P<sub>celA</sub> (100 base pairs combox promoter) was assembled with GFP reporter (BBa_E0240) using RFC10 format (BBa_K1379005). The construct BBa_K1379005 was then transformed into DH10B cells. To provide a positive control, BBa_I20260 containing strong constitutive promoter, strong RBS, and GFP generator was transformed into DH10B cells. Our negative control, BBa_K1379002, was the same as our experimental construct, but without the <i>SigmaX</i> gene.
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<p class="first_letter_enhanced">To test the functionality of &sigma;<sup>X</sup>, we first enable constitutive expression of &sigma;<sup>X</sup> in the &sigma;<sup>X</sup> Generator, <a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a>.
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The generator was then assembled with the standard promoter measurement kit <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>, with either promoter P<sub>celA</sub> (
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Promoter only: <a href= "http://parts.igem.org/Part:BBa_K1379000">BBa_K1379000</a>, w/ <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>:
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<a href= "http://parts.igem.org/Part:BBa_K1379002">BBa_K1379002</a>) and P<sub>comFA</sub> (Promoter only: <a href= "http://parts.igem.org/Part:BBa_K1379001">BBa_K1379001</a>,  
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w/ <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>: <a href= "http://parts.igem.org/Part:BBa_K1379003">BBa_K1379003</a>). <i>E. coli</i> colonies holding the resulting
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constructs in pSB3K3 were observed under fluorescent macroscope with UV filter. Measurement kit for standard reference promoter <a href= "http://parts.igem.org/Part:BBa_J23101">
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BBa_J23101</a>, which is <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
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</a> was used as the general negative control for background fluorescence. Measurement kits for P<sub>celA</sub> and P<sub>comFA</sub> without &sigma;<sup>X</sup> Generator were used
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as negative controls for function of &sigma;<sup>X</sup>.
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<p><br><b><u>Results</u></b> <br><br></p>
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<h5 style="font-size: 13px">Figure 1. P<sub>celA</sub> and P<sub>comFA</sub> promoters activated in presence of &sigma;<sup>X</sup>.</h5>
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<p><b>Results</b> <br><br>
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<h6 style= "font-size: 13px"> Only in the presence of &sigma;<sup>X</sup> would P<sub>celA</sub> and P<sub>comFA</sub> be turned on, as GFP expression could
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be seen when &sigma;<sup>X</sup> is present. Therefore, &sigma;<sup>X</sup> is functional.  P<sub>celA</sub> and P<sub>comFA</sub> gave little GFP signal in the
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<h5 style="font-size: 13px">Figure 1. P<sub>celA</sub> and P<sub>helicase</sub> promoter induced by SigmaX protein drives GFP expression.</h5>
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absence of &sigma;<sup>X</sup> but has comparable activity as reference promoter BBa_J23101 in presence of &sigma;<sup>X</sup>. Scale bar = 5mm.</h6>
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<h6 style= "font-size: 13px"> PcelA and Phelicase promoter induced by SigmaX protein gave GFP signals, while the same construct without SigmaX did not give any GFP signals. Another negative control which is only GFP generator without <i>sigmaX</i> and PcelA/Phelicase also did not give any GFP signals. Reference promoter J23100 + GFP is used as positive control. Scale bar = 5mm.</h6>
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<div class='content_1'><h3> <a href= "http://parts.igem.org/Part:BBa_K1379000">P<sub>celA</sub> (BBa_K1379000) </a></h3>
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<div class='content_1'><h3> <a href= "http://parts.igem.org/Part:BBa_K1379000">P<sub>celA</sub> (BBa_K1379000)</a> and <a href= "http://parts.igem.org/Part:BBa_K1379001">P<sub>comFA</sub> (BBa_K1379001) </a></h3>
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<p>To measure the R.P.U (Relative Promoter Unit) of PcelA 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 PcelA 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 <i>sigmaX</i> gene and PcelA promoter used in the construct are both cloned from E. coli NCTC 7465 strain. The experimental construct used was BBa_K1379005, which contain SigmaX protein generator, PcelA, 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 PcelA promoter with GFP generator but without ComX generator.<br>  </p>
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<p><br><br><u><b>Characterization Procedure</b></u><br><br>
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<p class="first_letter_enhanced"> <br><br>For characterization, P<sub>celA</sub> promoter was assembled with the promoter measurement kit <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> to give the P<sub>celA</sub> 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>. <br><br>
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1. Constructing BBa_K1379005-pSB3K3 (ComX Generator (BBa_K1379006)-PcelA-BBa_E0240-pSB3K3); Transforming BBa_K1379002-pSB3K3 (PcelA-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.<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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3. Culturing E. coli DH10B strain carrying BBa_K1379005-pSB3K3, BBa_K1379002-pSB3K3, BBa_I20260-pSB3K3, BBa_E0240-pSB3K3 in supplemented M9 medium and measuring the respective growth curve; <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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5. Calculating the Relative Promoter Units (RPU) using the obtained data; <br><br>
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P<sub>comFA</sub> promoter was assembled with the promoter measurement kit <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> to give the P<sub>comFA</sub> 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>. <br><br>
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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> and 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.
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<h5 style="font-size: 13px">Figure 2. PcelA promoter Relative Promoter Unit (RPU) is measured with reference to J23100 constitutive promoter.</h5>
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<h6 style= "font-size: 13px"> PcelA promoter induced by ComX protein 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 J23100 promoter strength. Measurement was done by using 3 replicas.</h6>
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    margin-right: auto" src= "https://static.igem.org/mediawiki/2014/8/8c/Wiki_hkust_pcelA_pcomFA.png"/><br>
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<h5 style="font-size: 13px">Figure 2. P<sub>celA</sub> has 0.53 RPU and P<sub>comFA</sub> hsa 1.21 RPU when paired with &sigma;<sup>X</sup> generator.</h5>
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<h6 style= "font-size: 13px"> P<sub>celA</sub> and 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> <br><br>
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<p><br><br><u><b>Data Processing</b></u><br><br>
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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); <br><br>
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2. For GFP intensity, GFP expression of BBa_K1379005-pSB3K3, BBa_K1379002-PSB3K3, and BBa_I20260-pSB3K3 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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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 PcelA and I20260 were calculated by dividing the GFP synthesis rate of BBa_K1379005-pSB3K3, BBa_K1379002-PSB3K3, and BBa_I20260-pSB3K3 over the average OD600 value; <br><br>
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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 PcelA absolute promoter activity over the averaged I20260 absolute promoter activity. R.P.U value of BBa_K1379005-pSB3K3 reflect the maximum GFP expression of PcelA promoter in the presence of ComX protein. Leakage could be analyzed according to the R.P.U value of BBa_K1379002-PSB3K3 which shows the GFP expression of PcelA promoter without ComX protein.<br><br>
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<div class='content_1'><h3><a href= "http://parts.igem.org/Part:BBa_K1379001"> P<sub>helicase</sub>(BBa_K1379001) </a> </h3>
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<div class='content_1'><h3>Characterization Method</h3>
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<p><u><b>Construction</b></u><br><br>
-
<img src= "https://static.igem.org/mediawiki/2014/archive/1/14/20141007050424!Phelicase_copy.png"/>
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1. Construct pSB3K3-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><br>
-
<h5 style="font-size: 13px">Figure 3. P<sub>helicase</sub> promoter Relative Promoter Unit (RPU) is measured with reference to J23100 constitutive promoter. </h5>
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<h6 style= "font-size: 13px"> P<sub>helicase</sub> promoter induced by ComX protein 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 J23100 promoter strength. Measurement was done by using 3 replicas.</h6>
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Or migrate <a href= "http://parts.igem.org/Part:BBa_K1379005">BBa_K1379005</a> to pSB3k3.<br><br>
-
</div>
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-
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2. Construct pSB3K3-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> <br>
 +
 
 +
Or migrate <a href= "http://parts.igem.org/Part:BBa_K1379007">BBa_K1379007</a> to pSB3K3.<br><br>
 +
 
 +
3. Transforming pSB3K3-P<sub>celA</sub>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br><br>
 +
 
 +
 
 +
4. Transforming pSB3K3-P<sub>comFA</sub>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br><br>
 +
 
 +
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>
 +
 
 +
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>
 +
 
 +
<p><u><b>Measurement</b></u><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>
 +
 
 +
2. Culturing <i>E. coli</i> DH10B strain carrying the whole construct listed on the construction part. 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>
 +
 
 +
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>
 +
 
 +
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>
 +
 
 +
6. Measuring the GFP intensity and OD595 values (we used Envision Multilabel Reader) every 30 minutes after the above mentioned <i>E. coli</i> strains are cultured to mid-log phase (OD600 = 0.3 - 0.5)<br><br>
 +
 
 +
Filter used on Envision Multilabel Reader: <br>
 +
- Absorbance :Photometric 595nm,<br>
 +
- Excitation :485nm FITC,<br>
 +
- Emission :535nm FITC, <br>
 +
- Mirror module : FITC (403) at bottom. <br><br>
 +
 
 +
- In between measurements, keep incubating the cells in 37°C while shaking. <br><br>
 +
 
 +
7. Calculating the Relative Promoter Units (RPU) using the obtained data; <br><br>
 +
</p>
 +
 
 +
 
 +
<p><br><u><b>Data Processing</b></u><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>
 +
 
 +
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>
 +
 
 +
 
 +
3. GFP synthesis rate was then obtained by calculating the slope of the above mentioned curve; <br><br>
 +
 
 +
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>
 +
 
 +
 
 +
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>
 +
Equation of the RPU calculation is shown below: <br>
 +
 
 +
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/e/e0/RPUequation_ust2014.png"/>
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<br><br>
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</p>
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<p>The method of measuring RPU (Relative Promoter Unit) of P<sub>helicase</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>helicase</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 ComX protein generator, P<sub>helicase</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>helicase</sub> promoter with GFP generator but without ComX generator.  </p>
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<u>References</u></p>
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<u><b>References</b></u><br><br></p>
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<p>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. <i>Journal of Biological Engineering</i>, 3, 4. doi: 10.1186/1754-1611-3-4</p>
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<p>
 +
 
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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
 +
<br><br>
 +
 
 +
Luo P., & Morrison D. (2003).<i> Transient Association of an Alternative Sigma Factor, ComX, with RNA Polymerase during the Period of Competence for Genetic Transformation in Streptococcus pneumoniae</i>. Journal of Bacteriology. doi:10.1128/JB.185.1.349-358.2003
 +
<br><br>
 +
 
 +
Piotrowski A., Luo P., & Morrison D. (2009). <i>Competence for genetic transformation in Streptococcus pneumoniae: termination of activity of the alternative sigma factor ComX is independent of proteolysis of ComX and ComW.</i> Journal of Bacteriology. doi:10.1128/JB.01750-08
 +
<br><br>
 +
 
 +
Rhodius V., Segall-Shapiro T., Sharon B., Ghodasara A., Orlova E., Tabakh H., . . . Voigt C. (2013). <i>Design of orthogonal genetic switches based on a crosstalk map of σs, anti-σs, and promoters.</i> Molecular Systhetic Biology .doi:10.1038/msb.2013.58
 +
<br><br>
 +
 
 +
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. <i>Journal of Biological Engineering</i>, 3, 4. doi: 10.1186/1754-1611-3-4
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Latest revision as of 22:28, 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 and 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 is 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 pSB3K3-σx Generator-(BBa_K1379006)-PcelA-BBa_E0240
Or migrate BBa_K1379005 to pSB3k3.

2. Construct pSB3K3-σx Generator-(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 the construction part. 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 (we used Envision Multilabel Reader) every 30 minutes after the above mentioned E. coli strains are cultured to mid-log phase (OD600 = 0.3 - 0.5)

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:





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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