Regulatory

Part:BBa_J23114:Experience

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

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Applications of BBa_J23114

Experience of the Nottingham 2018 iGEM team

Usage and Biology

BBa_J23114 is a constitutive promoter constructed by a previous iGEM team. We wanted to establish whether this promoter would be suitable for use in our non-model organism chassis Clostridium difficile, and also wanted to put the strength of this promoter into context by characterising fluorescence using the iGEM Interlab calibration curves, and also compare its strength to the Interlab positive and negative controls. The construct BBa_K2715114 allows us to characterise this promoter more extensively in E. coli with a standardised and reproducible measurement of fluorescence. Additionally this promoter was characterised in C. difficile using the gusA biobrick BBa_K330002 as a reporter gene in place of GFP, as GFP requires oxygen in order to function, and C. difficile is an anaerobic organism. The gusA containing composite used to assay the promoter activity in C. difficile is BBa_K2715026.

Characterisation

In these composite parts we've added a strong RBS BBa_K2715009, shown to function in Gram-positive and Gram-negative organisms, downstream of the BBa_J23114 promoter, driving expression of either GFP or GusA. The composites are part of a family of composite parts which were all characterised in the same plasmid backbone and in parallel in two fluorescence assays, the results of which can be seen below. The positive and negative controls are parts BBa_I20270 and BBa_R0040 respectively, used in the Interlab 2018 study. The composite parts tested in this assay under the same conditions using a range of alternative promoters are as follows:

GFP assay in E. coli

BBa_K2715106
BBa_K2715114
BBa_K2715119
BBa_K2715001
BBa_K2715002
BBa_K2715003
BBa_K2715004

BBa K2715114 family GFP assay 2.png

GusA assay in C. difficile The gusA containing composites tested in this assay under the same conditions using a range of alternative promoters are as follows:



BBa_K2715025
BBa_K2715026
BBa_K2715027
BBa_K2715028
BBa_K2715029
BBa_K2715030
BBa_K2715031

BBa K2715114 family gusA assay 2.png

Conclusions

These two composite parts have enabled a more standardised characterisation of BBa_J23114 when used in conjugation with a strong RBS shown to function in both Gram-positive and Gram-negative organisms, and the strength of BBa_J23114 can now be quantified using the iGEM 2018 interlab units of fluorescence, relative to the Interlab control plasmids, which will hopefully give future iGEM teams a more comprehensive understanding of its strength. We have also shown that this promoter has extremely low levels of activity in the Gram-positive organism C. difficile, and it’s therefore unlikely to be useful for other future iGEM teams looking to express genes in Gram-positive organisms.


Evaluation of Anderson promoter J23114 in B. subtilis by iGEM-Team LMU-Munich 2012

This Anderson promoter was evaluated without fused RFP with the lux operon as a reporter in B. subtilis. See the new BioBrick BBa_K823011 without RFP and have a look at the Data from the evaluation in B. subtilis.

User Reviews

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for iGEM-Team Goettingen 2012

Characterization experiment by qrtPCR on BBa_J23100, BBa_J23104, BBa_J23105, BBa_J23106, BBa_J23109, BBa_J23112, BBa_J23113, BBa_J23114 by iGEM Team Göttingen (by C. Krüger and J. Kampf)

Description

We used quantitative real-time PCR as a powerful tool for quantification of gene expression. We used this method to examine the expression rate of the Tar receptor gene under control of promoters from the Anderson family of the parts registry. The BioBricks (K777001-K777008) we used for this experiment can be found here.

The reported activities of these promoters are given as the relative fluorescence of these plasmids in strain TG1 [1]. Promoter constructs were cloned into the vector pSB1C3 and expressed in E.coli BL21DE3 grown in LB-media (lysogeny broth). The measurements were performed for each construct and reference as a triplet. Additionally, we included H2O as negative control to predict possible contamination. For the evaluation of our results, the 2–ΔΔCT (Livak) method was applied. We used the weakest promoter with the lowest expression rate as calibrator for the calculations and as reference the housekeeping gene rrsD of E.coli. You can find detailed information of the qrtPCR approach here.


Results & Discussion
Comparison of relative expression rates of constitutive promoters by qrtPCR and relative fluorescence (see parts registry,Anderson family). The blue bar indicates the measured expression rates for our constructs (J23100, J23104, J23105, J23106, J23109, J23112, J23113, J23114) and the red ones those for the literature values represented in the “parts registry”. The measurements are illustrated in a logarithmic application. The standard variation was calculated for our measured values (black error bar).

As mentioned before, both datasets were collected by methods which produce data at different points after the gene expression. Quantitative real time PCR measures the amount of expressed mRNA while relative fluorescence measurements quantify on protein level. In perspective of stability and half-life periods of mRNA and proteins or due to protein modification, it is comprehensible to obtain varying data-sets and expression rates. Another problem that occurred during our quantitative real-time measurements was the deviation in some of biological replicates. This problem was also observed in another group’s experiments (Kelly et al., 2009). They mentioned variations across experimental conditions in the absolute activity of the BioBricks. To reduce variation in promoter activity, they measured the activity of promoters relative to BBa_J23101. Furthermore, the iGEM team of Groningen which participated in 2009 also measured the relative fluorescence of TG1 strain with the promoters J23100, J23109 and J23106 via Relative Promoter Units (RPUs). Their values indicated the comparable tendency to our documented values
For a more detailed description of our results click here.


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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.

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.

Determination of Noise Levels in Constitutive Promoter Family Members

(Characterized by SDU-Denmark) 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.

For these experiments, the promoters controlling an RFP reporter system were cloned into E. coli strain MG1655 on pSB1A2. The cultures were grown in LB medium containing 50 µg/mL ampicillin and examined by fluorescence microscopy, using an Olympus IX83 with a photometrics prime camera and 200 ms exposure time, when cultures were at OD600=0.3-0.5. For flow cytometry, both LB medium with 50 µg/mL ampicillin and M9 medium with 100 µg/mL ampicillin were used. Excitation of RFP was at 561 nm, and emission was measured around 580 nm. 

The images obtained by fluorescence microscopy, which are given in Figure 1, revealed that the weak promoter, BBa_J23114, presented high variability in the expression of RFP between cells, indicating a high level of noise. Contrarily, RFP expression was more uniform in the bacterial cells containing the medium promoter Bba_J23106. Similarly, another medium strength promoter BBa_J23110, showed uniform level of RFP expression, although less consistent than the observed for BBa_J23106. Expectedly, the strong promoter, BBa_J23102 exhibited high RFP expression in cells and a low level of noise was observed.

T--SDU-Denmark--2.jpg

Figure 1.Fluorescence microscopy of RFP under the regulation of four members of the Anderson promoter collection, BBa_J23114, BBa_J23110, BBa_J23106 and BBa_J23102) on pSB1A2 in E. coli MG1655 at OD600=0.3-0.5. BBa_J23114 displayed a low and noisy expression of RFP, whereas both BBa_J23106 and BBa_J23110 showed a more uniform expression of medium intensity. The strongest promoter, BBa_J23102, exhibited a high level of consistent RFP expression.


The noise levels observed by fluorescence microscopy led to the design of an experiment, by which the cell population could be quantitatively studied. The change in expression levels and noise during growth of cell populations were tested for the weak and strong constitutive promoters, for which flow cytometry was used to assess the expression of the RFP reporter system. First, the cell populations were studied in LB media with 50 µg/mL ampicillin, thereby maintaining similar conditions as for the fluorescence microscopy experiment. All cultures were prepared from overnight cultures, obtaining a starting OD600=0.005.

T--SDU-Denmark--3.jpg

Figure 2. Flow cytometric fluorescence measurements in arbitrary units as a function of time. Left: Cultures were grown in LB medium. Right: Cultures were grown in M9 minimal medium supplemented with 0.2% glycerol. Fluorescence of RFP expressed by the weak and strong constitutive promoters were measured relative to the negative control WT E. coli MG1655. Samples where measured in technical replicates and standard error of mean is shown, but are in several cases indistinguishable from the graph.


The data obtained in this experiment revealed that the strong promoter displayed a higher basal level of RFP expression than the weak promoter. Moreover, both promoters exhibited similar decrease in fluorescence over time, as seen in Figure 2, indicating that a considerable portion of the cell populations for both promoters lost their ability to fluoresce during growth, with one population containing around 60% non-fluorescent bacteria, clearly divided from the fluorescent portion. As expected, the strong promoter displayed a high level of RFP expression, roughly 500-fold of the non-fluorescent MG1655 control at 1 hour. A 10-fold decrease in fluorescence was observed from 1 hour to 4 hours. The RFP expression mediated by the weak promoter exhibited a similar decrease in fluorescence over time, though with measured fluorescence levels 5 times lower than for the strong promoter.

Comparing these findings to the fluorescence microscopy data, it was hypothesised, that the increasing noise could be caused by bacterial plasmid loss. This could be ascribed to degradation of ampicillin in the medium, since the mechanism behind ampicillin resistance relies on the β-lactamase mediated cleavage of the antibiotic, thereby relieving the selective pressure. Consequently, the bacteria, which have lost their plasmid could be able to compete with the bacteria still containing their plasmid.
The fluorescence microscopy for the strong promoter did not exhibit a considerable level of noise, however, this does not reject the hypothesis. The increase in noise was revealed by flow cytometry, as measurements were carried out at several times throughout growth. However, the fluorescence microscopy measurements were only carried out once in the exponential phase, where the noise was not prevalent yet.
To substantiate the hypothesis, the experiment was performed anew using M9 minimal medium supplemented with 0.2 % glycerol and 100 µg/mL ampicillin, in an attempt to decrease the plasmid loss rate and optimise the selection.
The resulting data revealed, that the decrease in fluorescence levels had a lower rate for both promoters when the bacteria were cultured in M9 minimal medium, seen in the right graph in Figure 2, than in LB medium, seen in the left graph in Figure 2. Furthermore, when the bacteria were grown in M9 minimal medium with double amount of antibiotics compared to the LB medium, they displayed a higher level of fluorescence. Some of these observations could potentially be ascribed to the increased selection, that was strived after in this experiment. Moreover, the difference between the measured fluorescence levels for the two reporter systems was notably lower. This could indicate, that the reason behind the difference in expression levels between the weak and strong constitutive promoters, is due to the fact that bacteria cloned with the weak constitutive promoter are more prone to plasmid loss. However, it was observed that the bacteria expressing RFP under control of the weak promoter, did in fact fluoresce with a lower intensity.

To verify the hypothesis regarding the plasmid loss, the cultures were plated on 50 µg/mL ampicillin LA selection plates as well as LA plates without antibiotic, 12 hours after incubation start. For comparison, cultures containing either chloramphenicol and kanamycin resistance cassettes, were likewise plated out after similar treatment. The average plasmid loss measured as non-resistant CFU as a percentage of the total population is seen Figure 3.

T--SDU-Denmark--4.jpg

Figure 3. The average plasmid loss measured as non-resistant CFU as a percentage counts for the ampicillin resistance of the weak and strong promoter, as well as for chloramphenicol and kanamycin resistance controls. Samples were obtained for spread-plating at 12 hours after incubation start.


From this test, it is evident that antibiotic resistance is reduced for the ampicillin-resistance carrying bacteria, compared to chloramphenicol and kanamycin resistant cultures. This consolidates the hypothesis, that the bacteria transformed with the weak and strong promoter controlling RFP expression lose their plasmids throughout growth. However, it was further observed, that the plasmid loss in bacteria carrying the strong constitutive promoter was markedly higher than for bacteria carrying the weak constitutive promoter. This could be due to the fact that RFP in high concentrations is toxic for the cells, resulting in an increased pressure to lose the plasmid.

When using the promoter to express a gene, the observed plasmid loss should be taken into account, and compensated for, e.g. by continuous supply of antibiotic or substitution of the ampicillin resistance cassette.

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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: [2]

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.