Composite

Part:BBa_K733009

Designed by: WANG, Yuqi   Group: iGEM12_HKUST_Hong_Kong   (2012-09-16)

Ptms+BBa_E0240: Ptms+RBS+GFP+Double

Ptms is a promoter which proves to be functional in both E. coli and B. subtilis. 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 Link to our Regulation and Control Module

Promoter characterization 1.JPG

The key reason for using this low efficiency constitutive promoter in our construct is to enable our bacteria to express a low level of antitoxin so that the bacterial cell can only tolerate a certain amount of toxin. As the expression of BMP2 is tightly linked to the toxin, its expression can be regulated accordingly.


Objective

Our objective in characterizing this promoter is to test whether Ptms works in E. coli DH10B strain and determine its relative promoter unit (RPU) compared to the standard constitutive promoter (a promoter whose activity is arbitrarily valued at 1.0 by parts.igem.org).


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 using the absolute promoter activity as the final result, our characterization was based on obtaining the in vivo activity of this constitutive promoter. By adopting this method, we are able to eliminate errors caused by different experimental conditions and give a 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. At a time point around the mid-log phase, the GFP intensity and OD595 values 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/Reference Promoter) from the 2012 Distribution Kit

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 respective growth curve;

4. Measuring the GFP intensity and OD595 values every 15 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;

6. Compiling the results.


Data Processing 1. After E. coli carrying the right construct was grown to mid-log phase, GFP intensity and OD595 were measured every 15 minutes (up to 60min);

2. For GFP intensity, curve reflecting GFP expression change was plotted; for OD595, 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 Ptms and I20260 were calculated by dividing the corresponding GFP synthesis rate over the average OD595 value;

5. Averaged absolute promoter activity was then obtained by averaging the respective sets of absolute promoter activity values;

6. Finally, R.P.U was calculated by dividing the averaged Ptms absolute promoter activity over the averaged I20260 absolute promoter activity.


Result

1. Suggested by the GFP expression curve we plotted, Ptms functions in E.coli DH10B strain.

PTms GFP.jpg

* Above: GFP expression curve for one set of data

2. The RPU of Ptms obtained was 0.046497. This shows that Ptms has a very low promoter efficiency in E.coli DH10B strain.

PTms Promoter.jpg


Discussion

Compared to I20260, it seems that E. coli carrying Ptms-GFP has a rather low GFP expression. This may cause some difficulty in deciding whether Ptms functions in E. coli or not. However, referring to the curve (for GFP Intensity), the GFP expression for Ptms increased gradually with respect to time. This suggests that Ptms functions in E. coli DH10B strain, although with a low efficiency. A possible reason for its low efficiency could be that Ptms was originally from B. subtilis and was suggested to be functional in E. coli. Due to time constraints, we were unable to characterize the promoter in B. subtilis, and we still hope that we can address this in the future.

Reference

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.

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


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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 736



Functional Parameters: Austin_UTexas

BBa_K733009 parameters

Burden Imposed by this Part:

Burden Value: 0.0 ± 2.6%

Burden is the percent reduction in the growth rate of E. coli cells transformed with a plasmid containing this BioBrick (± values are 95% confidence limits). This BioBrick did not exhibit a burden that was significantly greater than zero (i.e., it appears to have little to no impact on growth). Therefore, users can depend on this part to remain stable for many bacterial cell divisions and in large culture volumes. Refer to any one of the BBa_K3174002 - BBa_K3174007 pages for more information on the methods, an explanation of the sources of burden, and other conclusions from a large-scale measurement project conducted by the 2019 Austin_UTexas team.

This functional parameter was added by the 2020 Austin_UTexas team.


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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