Difference between revisions of "Part:BBa K1379000"

Revision as of 20:27, 11 October 2014

P_celA

Usage and Biology

P_celA (or celAp) is a σ^X regulated promoter from Streptococcus pneumoniae. It is a member of the Cin-Box or Com-Box promoter family that share the 8 base pair consensus sequence of TACGAATA, where the sigma factor σ^X BBa_K1379004 (or ComX) binds to and promotes transcription initiation. During the exponential growth of S. pneumoniae, Competence Signal Peptide (CSP) mediated quorum sensing induces expression of σ^X. σ^X as a global regulator then directs S. pneumoniae to enter a transient competent cell state. P_celA, alongside with other Com-Box promoters, is turned on by σ^X and drives expression of the competence protein CelA.

Voigt and his colleagues have demonstrated that orthogonal gene expression could be achieved through the use of σs, anti-σs and synthetic promoters. (Rhodius et al., 2013). This principle has been demonstrated to work in E. coli (see characterization below), in which P_celA can only be turned on in the presence of σ^X.

iGEM 2014 Hong_Kong_HKUST Team has cloned P_celA from S. pneumoniae strain NCTC7465 and characterized its Relative Promoter Units (RPU) in presence and absence of a σ^X generator BBa_K1379006. Promoter P_comFA (BBa_K1379001), which is another Com-Box promoter recognized by σ^X, has also been characterized.

Characterization

For characterization, P_celA promoter was assembled with the promoter measurement kit BBa_E0240 to give the P_celA Measurement Kit BBa_K1379002 in plasmid pSB3K3. The construct was further assembled with σ^X generator BBa_K1379006 to give BBa_K1379005.

Qualitative characterization was performed by comparing intensities of GFP signals from colonies of E. coli holding the P_celA Measurement Kits with and without the σ^X 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.

Figure 1. P_celA is on in presence but not absence of σ^X. P_celA Measurement Kit gave strong GFP signals comparable to BBa_I20260 when assembled without σ^X generator, and fluorescence was not observed with the same construct without the σ^X generator. Scale bar = 5mm.

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 σ^X 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.

Figure 2. P_celA promoter Relative Promoter Unit (RPU) is measured with reference to BBa_J23101 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 BBa_J23101 promoter strength. Measurement was done by using 3 replicas.

Extensive documentation of this characterization, including the wet lab protocols and data processing, can be found in [http://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/characterization iGEM HKUST 2014 Wiki Page].

PcelA promoter is working in E. coli DH10B strain, and express GFP when induced with SigmaX protein. In comparison to the reference promoter 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.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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

@@ Line 12: / Line 12: @@
 ===Characterization===
-Characterization has been performed following protocol described in “Measuring the activity of BioBrick promoters using an <i>in vivo</i> reference standard” (Kelly et al., 2009). Briefly, P<sub>celA</sub> promoter was assembled  with the promoter measurement kit [[Part:BBa_E0240|BBa_E0240]] in plasmid [[Part:pSB3K3|pSB3K3]] with or without a &sigma;<sup>X</sup> generator [[Part:BBa_K1379006|BBa_K1379006]]. <i>E. coli</i> DH10B strains holding the two constructs respectively then grown to mid-log phase. 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 rate was then compared to that of standard reference promoter [[Part:BBa_J23101|BBa_J23101]] measurement device [[Part:BBa_I20260|BBa_I20260]] to obtain the Relative Promoter Units (RPUs). For subtraction of background fluorescence, pSB3K3 holding BBa_E0240 was measured alongside.
+For characterization, P<sub>celA</sub> promoter was assembled with the promoter measurement kit [[Part:BBa_E0240|BBa_E0240]] to give the P<sub>celA</sub> Measurement Kit [[Part:BBa_K1379002|BBa_K1379002]] in plasmid [[Part:pSB3K3|pSB3K3]]. The construct was further assembled with &sigma;<sup>X</sup> generator [[Part:BBa_K1379006|BBa_K1379006]] to give [[Part:BBa_K1379005|BBa_K1379005]].
-<br>
-[[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.]]
-<br><br>
+Qualitative characterization was performed by comparing intensities of GFP signals from colonies of <i>E. coli</i> 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 [[Part:BBa_J23101|BBa_J23101]],  [[Part:BBa_I20260|BBa_I20260]] was used as a positive control; [[Part:BBa_E0240|BBa_E0240]] was used as the negative control for background fluorescence.
+[[File:pcelAphoto1.png|600px|thumb|center|<b>Figure 1. P<sub>celA</sub> is on in presence but not absence of &sigma;<sup>X</sup>.</b> P<sub>celA</sub> Measurement Kit gave strong GFP signals comparable to [[Part:BBa_I20260|BBa_I20260]] when assembled without &sigma;<sup>X</sup> generator, and fluorescence was not observed with the same construct without the &sigma;<sup>X</sup> generator. Scale bar = 5mm.]]
+<br>
+Quantitative characterization was performed following the protocol described in “Measuring the activity of BioBrick promoters using an <i>in vivo</i> 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 [[Part:BBa_J23101|BBa_J23101]] measurement device [[Part:BBa_I20260|BBa_I20260]] to obtain the Relative Promoter Units (RPUs). For subtraction of background fluorescence, pSB3K3 holding BBa_E0240 was measured alongside.
+<br>
 [[File:pcelAgraph1.png|500px|thumb|center|'''Figure 2. P<sub>celA</sub> promoter Relative Promoter Unit (RPU) is measured with reference to [[Part:BBa_J23101|BBa_J23101]] 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.]]