Regulatory

Part:BBa_J23110:Experience

Designed by: John Anderson   Group: iGEM2006_Berkeley   (2006-08-17)

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Applications of BBa_J23110

User Reviews

The stochastic dynamics of the constitutive promoter family by 2013 Fudan iGEM team

We fused the constitutive promoters to a sfGFP reporter to test their stochastic dynamics by means of measuring fluorescence intensity of sfGFP using flow cytometry. In the meantime, we inserted the 16nt Csy4 insulator between the promoter and the reporter to stardardize the expression of the reporter downstream by eliminating the influence from 5'UTR. The following chart shows the distribution of sfGFP expression in the circumstance of promoter J23110.

Figure 1. The distribution of the sfGFP expression tested by flow cytometry with promoter J23110.
Figure 2. The intensity of different promoters is shown by the column height and the randomness of their functions by the bar width.

Evaluation of Anderson promoter J23110 in E. coli by iGEM Göttingen 2013



Shown here:
Upper two pictures: Growth curves of promoter strains on the left, growth curves of control strains on the right. Three biological replicates are shown.
Middle two pictures: RFP/OD600 of promoter strains on the left, RFP/OD600 of control strains on the right. Three biological replicates are shown.
Bottom three pictures: qRT PCR promoter analyses in three different growth phases. Promoters are normalised against BBa_J23117 .

Promotor 1:BBa_J23117
Promoter 2:BBa_J23116
Promoter 3:BBa_J23110
Promoter 4:BBa_J23118


The promoter strength was measured by using the reporter gene rfp.
Three different approaches were used: 1. RFP measurement, 2. qRT-PCR analyses and 3. single cell microscopy. Moreover, the first and the second approach characterised the promoter activity along the growth curve and to three important time points, respectively.
Most of our results from these approaches showed that BBa_J23110 might have the strongest promoter activity compared to BBa_J23116, BBa_J23117 and BBa_J23118. For more details, please read the discussion on our wikipage.


Fig. 1. RFP and qRT-PCR promoter analyses.


Fig. 2. Microscopic promoter analyses on single cell level
on the left side: bright field (BR), on the right side: RFP filter (rfp)
P1: BBa_J23117, P2: BBa_J23116, P3: BBa_J23110, P4: BBa_J23118, P8: control




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Wisconsin-Madison 2010

This constitutive promoter works very well. However, using it for molcular cloning is difficult. The level of expression acheived by J23100 caused our cells to grow slowly. After transformations we picked smaller colonies to get the best screens, and liquid cultures took an extra 4-6 hours to reach a decent plasmid prep OD. For future users, we recommend using it only at the end of your molecular cloning process to avoid the problems that arise from additional cellular stresses.

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iGEM Groningen 2009

We used a number of the constitutive promoter family members for testing our biobricks. The constitutive promoters show the expected level of fluorescence when transformed into E. coli TOP10 cells. Placing parts behind the promoters turned out to be relatively straight forward. We used this part in combination with several biobricks for building our constructs e.g. BBa_I750016 was placed behind the promoters.

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UNIPV-Pavia iGEM 2010

The BBa_J23100, BBa_J23101, BBa_J23105, BBa_J23106, BBa_J23110, BBa_J23114, BBa_J23116, BBa_J23118 were charcterized in LB and M9 supplemented with glycerol (0.4%) growth media in high copy and low copy vectors in E. coli TOP10 (BBa_V1009).

RPU and doubling time were characterized for all of them, according to the protocols reported below.

The following measurement systems were used for high copy plasmids:

In order to build low copy plasmid measurement systems, the EcoRI-PstI fragment (J231xx-RFP) of each BBa_J61002-BBa_J231xx was assembled into pSB4C5 vector. This fragment contains the constitutive promoter of interest upstream a RBS-RFP-TT expression system.

The following measurement parts were used for low copy plasmids:


The RPU values and doubling times are here reported:

Figure 5 - R.P.U. of the studied promoters from Anderson promoters' collection, LB medium and high copy plasmid (BBa_J61002)
Figure 6 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and high copy plasmid (BBa_J61002)
Figure 7 - R.P.U. of the studied promoters from Anderson promoters' collection, M9 medium and low copy plasmid (pSB4C5). These plasmids were constructed by assembling the EcoRI-PstI fragment of BBa_J61002-BBa_J231xx in pSB4C5 vector, in order to transfer the promoter and the RBS-RFP-TT expression construct from BBa_J61002 to pSB4C5.

The error bars represent the standard deviation for three dfferent wells in the same experiment. Doubling times were evaluated for the described cultures (HC stands for High Copy and LC stands for Low Copy):

Promoter doubling time [minutes]
LB in HC plasmid M9 in HC plasmid M9 in LC plasmid
BBa_J23100 33.75
±
1.34
82.53
±
2.45
86.11
±
4.45
BBa_J23101 35.93
±
0.62
82.68
±
1.84
86.42
±
1.91
BBa_J23105 29.86
±
0.33
63.09
±
7.08
85.00
±
5.13
BBa_J23106 29.17
±
0.96
68.11
±
4.25
88.71
±
0.90
BBa_J23110 31.28
±
0.42
67.52
±
5.87
76.15
±
2.16
BBa_J23114 28.97
±
0.49
59.44
±
5.20
80.12
±
0.95
BBa_J23116 28.14
±
0.25
72.74
±
0.37
81.68
±
3.08
BBa_J23118 32.84
±
0.31
73.64
±
2.41
89.86
±
2.93

It was not possible to evaluate promoters activities in low copy number plasmids in LB because the RFP activity was too weak and not distinguishable from the background.

Discussion: we observed that the ranking previously documented in the Registry is not valid in all the tested conditions, even if a general agreement can be observed. As an example, BBa_J23110 in high copy plasmid is stronger than BBa_J23118, in contrast with the ranking reported in the Registry.

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Uppsala University 2012

iGEM Team Uppsala University 2012

Promoter strength

Promoter strenghts in MG1566 and DH5alpha, relative to J23101.

A promoter test was carried out to put synthetic and natural promoters on the same scale. Every promoter was assembled before B0032-SYFP2 (BBa_K864101) in the backbone BBa_K592200 (very similar to the pSB3x5 backbones). The test was performed in E coli expression strain MG1655 and cloning strain DH5alpha, by flow cytometry fluorescence measurements of single cells.

Triplicates of each strain and promoter were inoculated in 2 mL LB media with spectinomycin (50 µg/mL) and grown overnight shaking at 37° C. Samples were equilibrated in PBS solution at 1:160 dilution for one hour, and then measured by a BD Biosciences FACSaria III. 10^5 cells of each sample were individually measured and averaged, with dead and other non-flourescent cells excluded. Promoter strength is noted as fractions of the reference promoter's, J23101, strength in corresponding strain.

MG1566 DH5alpha
J23101 1 1
J23106 0.19 0.37
J23110 0.27 0.50
J23116 N/A 0.11
Plac 0.34 0.67
PlacIq 0.03 0.05
T5lac 0.217 0.54
PLlacO 0.87 N/A

The variance in expression between MG1655 and DH5α may depend on the reference J23101. The maximum protein expression may be lower in DH5α, due to its lower fitness resulting in lower expression of SYFP2 in the J23101 construct. Alternatively, the clone with J23101 in DH5α may have been weaker than average, resulting in higher RPU values compared to other DH5α.

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Microplate reader experiments for constitutive promoters (R.P.U. evaluation)

  • 8 ul of long term storage glycerol stock were inoculated in 5 ml of LB or M9 + suitable antibiotic in a 15 ml falcon tube and incubated at 37°C, 220 rpm for about 16 hours.
  • The grown cultures were then diluted 1:100 in 5 ml of LB or M9 supplemented medium and incubated in the same conditions as before for about 4 hours.
  • These new cultures were diluted to an O.D.600 of 0.02 (measured with a TECAN F200 microplate reader on a 200 ul of volume per well; it is not equivalent to the 1 cm pathlength cuvette) in 2 ml (wanted final volume) LB or M9 + suitable antibiotic. In order to have the cultures at the desired O.D.600 (O.D._wanted=0.02), the following dilution was performed:
UNIPV Pavia OD600 dil.png
  • These new dilutions were aliquoted in a flat-bottom 96-well microplate, avoiding to perform dynamic experiments in the microplate frame (in order to prevent evaporation effects in the frame). All the wells were filled with a 200 ul volume.
  • The microplate was incubated in the Tecan Infinite F200 microplate reader and fluorescence and absorbance were measured with this automatic protocol:
    • 37°C constant for all the experiment;
    • sampling time of 5 minutes;
    • fluorescence gain of 50 or 70;
    • O.D. filter was 600 nm;
    • GFP filters were 485nm (ex) / 540nm (em);
    • RFP filters were 535nm (ex) / 620nm (em);
    • 15 seconds of linear shaking (3mm amplitude) followed by 10 seconds of waiting before the measurements in order to make a homogeneous culture.
    • Experiment duration time: about 6 hours.


Data analysis for RPU evaluation

The RPUs are standard units proposed by Kelly J. et al., 2009, in which the relative transcriptional strength of a promoter can be measured using a reference standard.

RPUs have been computed as:

UNIPV Pavia RPU formula.png

in which:

  • phi is the promoter of interest and J23101 is the reference standard promoter (taken from the Anderson Promoter Collection);
  • F is the blanked fluorescence of the culture, computed by subtracting for each time sample the fluorescence value of a negative control (a non-fluorescent culture). In our experiments, the TOP10 cells bearing BBa_B0032 or BBa_B0033 were usually used because they are RBSs and do not have expression systems for reporter genes;
  • ASB is the blanked absorbance (O.D.600) of the culture, computed as described in "Preliminary remarks" section.

RPU measurement has the following advantages (under suitable conditions)

  • it is proportional to PoPS (Polymerase Per Second), a very important parameter that expresses the transcription rate of a promoter;
  • it uses a reference standard and so measurements can be compared between different laboratories.

The hypotheses on which RPU theory is based can be found in Kelly J. et al., 2008, as well as all the mathematical steps. From our point of view, the main hypotheses that have to be satisfied are the following:

  • the reporter protein must have a half life higher than the experiment duration (we use GFPmut3 - BBa_E0040 -, which has an estimated half life of at least 24 hours, or an engineered RFP - BBa_E1010, for which the half life has not been measured, but is qualitatively comparable with the GFP's);
  • strain, plasmid copy number, antibiotic, growth medium, growth conditions, protein generator assembled downstream of the promoter must be the same in the promoter of interest and in J23101 reference standard.
  • steady state must be valid, so (dF/dt)/ASB (proportional to the GFP synthesis rate per cell) must be constant.

In order to compute the RPUs, the Scell signals ((dGFP/dt)/ASB)) of the promoter of interest and of the reference J23101 were averaged in the time interval corresponding to the exponential growth phase. The boundaries of exponential phase were identified with a visual inspection of the linear phase of the logarithmic growth curve. |} UNIQ696d8443e0b2c2e2-partinfo-00000046-QINU


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iGEM Uppsala 2015

iGEM Uppsala 2015 review

The results below is from Uppsala iGEM team 2015

The constitutive promoters J23101 and J23110 have been used extensively in our project.

In this iGEM project we faced the need to characterize a promoter, the NahR/Psal system (Schell, Mark A. "Homology between nucleotide sequences of promoter regions of nah and sal operons of NAH7 plasmid of Pseudomonas putida.", 1986) which is induced by salicylate. We decided to compare it with different amounts of salicylate concentrations and also with two constitutive promoters (the J23101 and J23110). Among the existing approaches for measuring the strength of a promoter, we chose to use fluorometry. We did not have access to a fluorometer in our lab, so we decided to build our own.

To test the reliability of our constructed fluorometer, we wanted to compare the measurement with an existing method for fluorometry. MACS, Magnetic-Activated Cell Sorting is a method where the fluorescence of individual cells in a culture can be measured. When taking the average fluorescence of a large number of cells, the mean fluorescence of the culture can be deduced. Four individual colonies of DH5α containing the constitutive Anderson promoter J23110 and dTomato ( BBa_K1688011) on a pSB1K3 plasmid were transferred to overnight cultures as biological replicates. These were then diluted 1:300 in PBS buffer and measured on a MACSQuant VYB, Miltenyi Biotec. Excitation wavelength was set at 561 nm, with a 615/20 nm band pass filter for the emission. Parallel measurements were performed on the same biological replicates (not diluted) using our fluorometer.

Uppsala_fluoro_5.png

Figure 2: Fluorescence of dTomato under J23110 constitutive promoter in pSB1K3 in DH5α. Measurement was performed using MACSQuant VYB (excitation 561 nm, 615/20 nm band pass filter) and our fluorometer (excitation 554 nm, long pass filter 590 nm). Fluorometer order of magnitude: 25. The error bars show the maximum difference between the measurements, within each method.

The Anderson promoters were also used for the expression of our biosurfactants. The bacteria Pseudomonas aeruginosa is one of the best characterized organisms that produces rhamnolipids, which are biosurfactants (Oschner et al. 1995). The enzymes responsible for producing the mono-rhamnolipids are called RhlA and RhlB. These make up the subunits of rhamnosyltransferase I and are organized in an operon for highest efficiency. The drop collapse test, CTAB test, TLC and mass spectrometry showed positive result and we could confirm that mono-rhamnolipids are expressed by our construct (BBa_K1688000) with E.coli BL21DE3. The promoters successfully expressed our biosurfactants

Determination of Noise Levels in Constitutive Promoter Family Members

(characterized by SDU-Denmark 2017)

Fluorescence microscopy and flow cytometry revealed decrease in fluorescence over time for members of the constitutive promoter family.
The expression levels and the noise of four different members of the Anderson promoter collection and their RFP reporter systems, were studied by fluorescence microscopy. These were, in increasing promoter strength, BBa_J23114, BBa_J23110, BBa_J23106, and BBa_J23102
Additionally, the change in RFP expression levels and noise during growth were tested for the promoters with the highest and lowest relative promoter strength by flow cytometry and qualitative analysis by fluorescence microscopy. Combining these two techniques, the expression and noise levels for the promoters were determined as follows:
- The weak promoter, BBa_J23114, exhibited a relatively low expression of RFP, indicating low gene expression and an increasing high level of noise throughout growth.
- Both medium strength promoters, BBa_J23110 and BBa_J23106, displayed a moderate level of both noise and protein expression of the RFP reporter.
- The strong promoter, BBa_J23102, exhibited a comparatively high expression of the reporter RFP and an increasing high level of noise throughout growth.


Determination of Noise Levels in Constitutive Promoter Family Members

(characterized by SDU-Denmark 2017)

Fluorescence microscopy and flow cytometry revealed decrease in fluorescence over time for members of the constitutive promoter family.
The expression levels and the noise of four different members of the Anderson promoter collection and their RFP reporter systems, were studied by fluorescence microscopy. These were, in increasing promoter strength, BBa_J23114, BBa_J23110, BBa_J23106, and BBa_J23102
Additionally, the change in RFP expression levels and noise during growth were tested for the promoters with the highest and lowest relative promoter strength by flow cytometry and qualitative analysis by fluorescence microscopy. Combining these two techniques, the expression and noise levels for the promoters were determined as follows:
- The weak promoter, BBa_J23114, exhibited a relatively low expression of RFP, indicating low gene expression and an increasing high level of noise throughout growth.
- Both medium strength promoters, BBa_J23110 and BBa_J23106, displayed a moderate level of both noise and protein expression of the RFP reporter.
- The strong promoter, BBa_J23102, exhibited a comparatively high expression of the reporter RFP and an increasing high level of noise throughout growth.

T--SDU-Denmark--2.jpg


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University of Texas at Austin iGEM 2019

UT Austin iGEM 2019: Characterization of metabolic burden of the Anderson Series

Description

The 2019 UT Austin iGEM team transformed the Anderson Series promoters into our 'burden monitor' DH10B strain of E. coli, which contains a constitutive GFP cassette in the genome of the cell. GFP expression fluctuates depending on the number of ribosomes available. Using this strain, we characterized the relative burden (percent reduction in growth rate) of each Anderson Series part. Our results showed a range of growth rate reductions for each of these parts due to ribosomal reallocation from the genome of the host cell, towards the expression of RFP. Anderson Series parts with strong promoters are depicted with darker red colors and Anderson Series parts with weak promoters are depicted with lighter pink colors to show relative RFP expression. We saw a positive correlation between relative promoter strength and metabolic burden; parts with stronger promoters expressed less GFP and had a lower growth rate than parts with weaker promoters. The regression line for the graph below was constructed by measuring the burden of 5 parts that were created by the 2019 UT Austin iGEM team that each contained an Anderson Series promoter (BBa_J23104 or BBa_J23110), an RBS of varying strength, and a BFP reporter. For more information on characterization of these parts through the burden monitor, visit our team’s wiki page: [1]

Fig.1:Growth vs GFP Expression graph showing the relative burden positions of the Anderson Series promoters. The parts with strong promoters are depicted in dark red and are clustered near the bottom of the graph because they have lower growth rates and express lower levels of GFP as a result of high cellular burden. The parts with weaker promoter are depicted in light pink ad are clustered near the top of the graph because they have higher growth rates and express higher levels of GFP as a result of low cellular burden.


Table.1: Burden measurements for the Anderson Series promoters measured as percent reduction in growth rate ± 95% confidence interval.

Importance of Characterizing Burden

Although often we cannot avoid using a specific burdensome part, knowing in advance that it is burdensome, and that it has a high chance of mutating into a non-functional genetic device, can help with troubleshooting and coming up with alternatives. In the specific case of fluorescent protein-expressing devices, Fluorescence-activated cell sorting (FACS) can be used to filter out individual cells that meet a certain fluorescence threshold. This way, the cells expressing lower levels of the fluorescent protein are weeded out of the population.