View source for Part:BBa K1379000

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<partinfo>BBa_K1379000 short</partinfo>

===Usage and Biology===

P<sub>celA</sub> (also known as <i>celAp</i>) is a promoter in <i>Streptococcus pneumoniae</i> under the regulation of sigma factor &sigma;<sup>X</sup>.

A sigmaX-inducible promoter initiating the transcription of competence CelA protein in ''Streptococcus pneumoniae'' NCTC7465 strain. PcelA is a promoter which proves to be functional in ''E. coli'' and ''S. pneumonia''. This promoter works when induced by SigmaX protein [[Part:BBa_K1379004|BBa_K1379004]]. The sigmaX protein will bind to 8 base pairs of PcelA promoter and trigger gene expression. The sigmaX gene and PcelA promoter used in the construct are both cloned from ''S. pneumoniae'' NCTC 7465 strain. R.P.U (Relative Promoter Unit) of PcelA is measured to represent promoter strength in reference to constitutive promoter [[Part:BBa_J23100|BBa_J23100]]. PcelA promoter is characterized in E. coli DH10B cell as it is frequently used by iGEM participants. 
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==Characterization==

== Method ==

By linking PcelA promoter with GFP generator ([[Part:BBa_E0240|BBa_E0240]]), and SigmaX generator([[Part:BBa_K1379006|BBa_K1379006]]), the promoter activity was represented by the GFP synthesis rate. Fluorescence was measured 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.
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To measure the RPU (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). With the use of EnVision multilabel reader from Perkin Elmer Company, it enabled us to obtain the fluorescence and absorbance of cells over time. GFP intensity and OD595 values were measured every 30 minutes after the E. coli strains are cultured to mid-log phase.
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The positive control used in this characterization is [[Part:BBa_I20260|BBa_I20260]] which is a constitutive promoter [[Part:BBa_J23100|BBa_J23100]] containing GFP generator [[Part:BBa_E0240|BBa_E0240]], while the negative control used in this characterization is [[Part:BBa_K1379002|BBa_K1379002]] which is PcelA promoter with GFP generator but without SigmaX generator. 
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The detailed description including characterization procedure and Data processing of our characterization can be found in [http://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/characterization iGEM HKUST 2014 Wiki Page].

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[[File:pcelAphoto1.png|600px|thumb|center|'''Figure 1. PcelA promoter induced by SigmaX protein drives GFP expression. While the same construct without SigmaX protein did not give any GFP signals. Another negative control which is only protein sigmaX without PcelA also did not give any GFP signals. Reference promoter [[Part:BBa_J23101|BBa_J23101]] + GFP is used as positive control. Scale bar = 5mm.]]

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[[File:pcelAgraph1.png|500px|thumb|center|'''Figure 2. PcelA promoter Relative Promoter Unit (RPU) is measured with reference to [[Part:BBa_J23100|BBa_J23100]] constitutive promoter. PcelA promoter induced by SigmaX 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 [[Part:BBa_J23101|BBa_J23101]] promoter strength. Measurement was done by using 3 replicas.]]
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== Discussion ==

PcelA promoter is working in ''E. coli'' DH10B strain, and express GFP when induced with SigmaX protein. In comparison to the reference promoter [[Part:BBa_J23101|BBa_J23101]], the R.P.U (Relative Promoter Unit) of PcelA promoter is around 0.5. From Figure 2, we could tell that this promoter is specific as there is almost no GFP expression in the absence of SigmaX protein inducer. This might indicate that the 8 basepairs combox region in PcelA promoter is specific to SigmaX. However, further characterization needs to be done to evaluate crosstalks between this inducer-promoter system.
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<span class='h3bb'>Sequence and Features</span>
<partinfo>BBa_K1379000 SequenceAndFeatures</partinfo>


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===Functional Parameters===
<partinfo>BBa_K1379000 parameters</partinfo>
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==Reference ==
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<br><br><br>