Team:Hong Kong HKUST/pneumosensor/characterization

From 2014.igem.org

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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>E. coli</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">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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2. Preparing supplemented M9 medium (M9 Minimal salt medium protocols could be seen on the protocols page) <br><br>
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 <i>Streptococcus pneumoniae</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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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>
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>
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>

Revision as of 15:00, 10 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)

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 σx gene and PcelA promoter used in the construct are both cloned from Streptococcus pneumoniae NCTC 7465 strain. The experimental construct used was BBa_K1379005, which contain σx 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 σx generator.

Figure 2. PcelA promoter Relative Promoter Unit (RPU) is measured with reference to J23101 constitutive promoter.
PcelA promoter induced by σx 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.

PcomFA(BBa_K1379001)

Figure 3. PcomFA promoter Relative Promoter Unit (RPU) is measured with reference to J23101 constitutive promoter.
PcomFA promoter induced by σx 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.

The method of measuring RPU (Relative Promoter Unit) of PcomFA promoter is similar to measuring RPU of PcelA 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 PcomFA and PcelA. Fluorescence and absorbance are measured in the same time period. The experimental construct used was BBa_K1379007, which contain σx generator, PcomFA, 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 PcomFA promoter with GFP generator but without σx generator.

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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