Part:BBa_K733009
Ptms+BBa_E0240: Ptms+RBS+GFP+Double
Ptms is a promoter which proves to be functional in both E. coli and B. subilis. R.P.U standard is advocated by part registry to represent the efficiency of a constitutive promoter.(Kelly et al., 2009) We intend to characterize promoter Ptms in E. coli. Using E. coli as our initial characterization chassis may prove to be more valuable for most of iGEM participants, because E. coli is frequently used by iGEM participants.
Link to our Ptms promoter: BBa_K733001
Characterization
Background Information [http://2012.igem.org/Team:HKUST-Hong_Kong/Module/Regulation_and_control Link to our Regulation and Control Module]
The basic reason of using this low efficiency constitutive promoter is to enable our bacteria to express a low level of antitoxin so that a certain amount of toxin can be balanced; thus the Bmp2 whose expression is tightly linked with the toxin can be expressed at a certain level as well.
Objective
Our objective for characterizing this promoter is to test whether Ptms works in E.coli DH10B strain and determine the relative promoter units (RPU) of it to the standard constitutive promoter activity reference point given by part registry so that we and the subsequent IGEM teams may have an idea how efficient this promoter is.
Intended Result
1. Ptms should work in E.coli. This is supported by previous research (Moran et al., 1982).
2. The activity of Ptms should be relatively low.
Method
Instead of measuring the absolute promoter activity, our characterization was generally based on measuring the relevant in vivo activity of this constitutive promoter. By adopting this method, we may be able to eliminate the error caused by different experimental conditions and give a relatively more convincing result. By linking the promoter with GFP (BBa_E0240), the promoter activity was represented by the GFP synthesis rate which can be easily measured. E.Coli carrying the right construct was then cultured to log phase. During a time slot around the mid-log phase, the GFP intensity and OD595 value were measured to obtain the Relative Promoter Units (RPU).
Characterization Procedure
1. Constructing BBa_K733009-pSB3K3 (Ptms-BBa_E0240-pSB3K3); Transforming BBa_I20260-pSB3K3 (Standard Constitutive Promoter Activity Reference Point) from 2012 Distribution;
2. Preparing supplemented M9 medium (see below);
3. Culturing E.coli DH10B strain carrying BBa_K733009-pSB3K3 and E.coli carrying BBa_I20260-pSB3K3 in supplemented M9 medium and measuring the growth curve respectively;
4. Measuring the GFP intensity and ODA595 value every 15 minutes after E.coli carrying BBa_K733009-pSB3K3 and E.coli carrying BBa_I20260 are cultured to mid-log phase;
5. Calculating the Relative Promoter Unites (RPU) using the obtained data;
6. Compiling the result.
Data Processing
1. After E.coli carrying the right construct was grown into mid-log phase, GFP intensity and ODA595 were measured every 15 minutes (up to 60min);
2. For GFP intensity, curve reflecting GFP expression change was plotted; for ODA595, average values was taken;
3. GFP synthesis rate was then represented by the slope of the curve reflecting GFP expression change;
4. Absolute promoter activity of Ptms and I20260 were calculated by divide the corresponding GFP synthesis rate by the average ODA595 value;
5. Absolute promoter activity was then modified by taking the average value of all sets of data obtained;
6. Finally, R.P.U was calculated by dividing Ptms absolute promoter activity by I20260 absolute promoter activity.
Result
1. Suggested by the GFP expression curve we plotted, Ptms functions in E.coli' DH10B strain.
* GFP expression curve for one set of data
2. The overall RPU was calculated as 0.046497. It has been shown that Ptms has a very low promoter efficiency in E.coli DH10B stain.
Discussion
Compared with I20260, it seems that bacteria carrying Ptms had a rather low GFP expression. This may cause some difficulty upon deciding whether Ptms functions in E.coli or not. However, since viewed from the curve, the GFP expression for Ptms increased gradually in respect to time. These all give us good reason to say that Ptms functions in E.coli DH10B strain, although with a low efficiency. Another reason for its low efficiency could be that Ptms was originally got from B.subtilis and is only suggested to be functional in E.coli. While we haven’t got time to characterize the promoter in B.subtilis, we still hope that in the future we can actually achieve that.
Supplemented M9 Medium Composition
1. 5X M9 Salt Composition (1L)
(1) 64g Na2HPO4﹒
(2) 15g KH2PO4
(3) 2.5g NaCl
(4) 5.0g NH4CL
2. Minimal 1X M9 medium (1L)
(1) 200ml of 5X M9 Salt
(2) 2ml of 1M MgSO4
(3) 100μl of 1M CaCl2
(4) 5ml of 40% glycerol
3. Supplement (for the final medium)
(1) 1mM thiamine hydrochloride
(2) 0.2% casamino acids
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 736
Reference
Kelly J, Rubin A, Davis J, Ajo-Franklin C, Cumbers J, Czar M, de Mora K, Glieberman A, Monie D, Endy D: Measuring the activity of BioBrick promoters using an in vivo reference standard. Journal of Biological Engineering 2009, 3:4.
Moran, C., Lang, N., LeGrice, S., Lee, G., Stephens, M., Sonenshein, A., et al. (1982). Nucleotide sequences that signal the initiation of transcription and translation in Bacillus subtilis..Molecular and General Genetics MGG,186, 339-346.
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