Part:BBa_J23118:Experience

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Applications of BBa_J23118

Evaluation of Anderson promoter J23118 in E. coli by [http://2013.igem.org/Team:Goettingen 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_J23118 might have a lower promoter activity compared to BBa_J23110, but a higher promoter activity compared to BBa_J23117 and BBa_J23116. 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

Evaluation of Anderson promoter J23118 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_K823014 without RFP and have a look at the [http://2012.igem.org/Team:LMU-Munich/Data/Anderson Data] from the evaluation in B. subtilis.

Characterization

The activity of the promoter was checked in TOP10 cells, which are negative for the tet repressor, and also in DH5 alpha pro cells which have tet repressor gene integrated into the genome. The red fluorescence was measured by FACs with different inducer concentrations in both the cell types. It was observed that under all the inducer concentrations the promoter activity remains the same. It was reported to have 2 fold higher activity than the J23105.

Fluorescence intensities of RFP with weak promoter

Fluorescence intensities of RFP with strong promoter

Comparison of strong and weak promoters

Characterization experiment on BBa_J23100, BBa_J23101, BBa_J23118 - UNIPV-Pavia Team (Test performed by L.Pasotti, S.Zucca, E. Del Fabbro)

Description

These three promoters are from the Anderson Promoter Collection, which is a library of constitutive sigma70 bacterial promoters. The strength of each promoter of the library has already been estimated in saturation growth phase cultures in LB, but here we provide the characterization of BBa_J23100 and BBa_J23118 in standard units (RPUs) in LB medium, in order to add experience and data for these BioBricks. BBa_J23101 is the reference standard promoter, so it has RPU=1 for definition.

The data shown below are referred to BBa_K173000, BBa_K173001 and BBa_K173002 that are the measurement parts of BBa_J23100, BBa_J23101 and BBa_J23118 respectively.

Characterization

Compatibility: E. coli TOP10 in pSB1A2

Part	LB
Part	Doubling time [minutes]	RPU
BBa_J23100 (in BBa_J61002 plasmid)	36	not computed
BBa_J23101 (in BBa_J61002 plasmid)	37	not computed
BBa_J23118 (in BBa_J61002 plasmid)	36	not computed
BBa_K173000	36	2.04 [1.99 ; 2.08]
BBa_K173001	36	1 (reference standard)
BBa_K173002	35	0.69 [0.64 ; 0.73]

BBa_J231xx Growth curves in LB

BBa_J231xx (dGFP/dt)/OD

Growth conditions

Microplate reader experiments

8 ul of long term storage glycerol stock were inoculated in 5 ml of LB + 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 comparable with the 1 cm pathlength cuvette) in a sufficient amount of medium to fill all the desired microplate wells.
These new dilutions were aliquoted in a flat-bottom 96-well microplate, avoiding to perform dynamic experiments in the microplate frame (see [http://2009.igem.org/Team:UNIPV-Pavia/Methods_Materials/Evaporation Frame effect section] for details). All the wells were filled with a 200 ul volume.
If required, 2 ul of inducer were added to each single well.
The microplate was incubated in the Tecan Infinite F200 microplate reader and fluorescence (when required) and absorbance were measured with this automatic protocol:
- 37°C constant for all the experiment;
- sampling time of 5 minutes;
- fluorescence gain of 50;
- O.D. filter was 600 nm;
- GFP filters were 485nm (ex) / 540nm (em);
- 15 seconds of linear shaking (3mm amplitude) followed by 10 seconds of waiting before the measurements in order to make a homogeneous culture.
- Variable experiment duration time (from 3 to 24 hours).

Data analysis

Growth curves

All our growth curves have been obtained subtracting for each time sample the broth O.D.600 measurement from that of the culture; broth was considered in the same conditions of the culture (e.g. induced with the same inducer concentration).

Doubling time

The natural logarithm of the growth curves (processed according to the above section) was computed and the linear phase (corresponding to the bacterial exponential growth phase) was isolated by visual inspection. Then the linear regression was performed in order to estimate the slope of the line m. Finally the doubling time was estimated as d=ln(2)/m [minutes].

In the case of multiple growth curves for a strain, the mean value of the processed curves was computed for each time sample before applying the above described procedure.

Relative Promoter Units (RPUs)

The RPUs are standard units proposed by Kelly J. et al., 2008, in which the transcriptional strength of a promoter can be measured using a reference standard, just like the ground in electric circuits.

RPUs have been computed as:

in which:

phi is the considered promoter and J23101 is the reference standard promoter (taken from Anderson Promoter Collection);
F is the blanked fluorescence of the culture, computed subtracting for each time sample fluorescence measure for negative control from that of culture, where the negative control is a non-fluorescent strain (in our experiment it is usually used TOP10 strain bearing BBa_B0032 or BBa_B0033, which are symmply RBSs do not have expression systems for reporter genes);
ABS is the blanked absorbance (O.D.600) of the culture, computed as described in "Growth curves" 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_E0240, which has an estimated half life of at least 24 hours, and the experiments duration is always less than 7 hours);
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)/ABS (proportional to the GFP synthesis rate per cell) must be constant.

Inducible systems

Every experiment is performed on the following cultures:

the culture of interest (system studied expressing GFP)
the benchmarck used to evaluate R.P.U. (BBa_K173001 measurement part, that is BBa_J23101 with BBa_E0240 downstream)
a negative control (generally, BBa_B0033 RBS)

For inducible systems several plots are reported. The first plot is a panel containing 4 subplots, numerated this way:

(1)	(2)
(3)

Plot (1) contains growth curves of the cultures, after blank value has been removed. Every curve is calculated averaging on three replicates of the same culture and subtracting the blank for each time sample. Blank is calculated averaging the replicates of blank wells.

Plot (2) shows the logarithm of absorbance in exponential phase of bacterial growth, determined by a visual inspection of log-plots. These values are used to evaluate doubling time and R.P.U..

Plot (3) contains (dGFP/dt)/O.D., the value named S_cell in Kelly J. et al., 2008 procedure for RPU evaluation.

In these plots are reported black veritcal lines that define the range of values used to evaluate RPU. It is important to underline, as explained in next paragraph, that RPU are calculated on cultures at the same O.D. level, not at the same time.

The second graphic shows S_cell VS O.D.. This plot allows the conparison of S_cell values between different cultures, that are supposed to reach the same level of growth not at the same time, but at the same O.D. value.

The third graphic shows the induction curve. The RPU value is calculated on S_cell values corresponding to O.D. values in exponential phase (typically, from 0.05 to 0.16). The curve is obtained averaging in time S_cell values corresponding to exponential phase.

Error bars rapresent the minimum and maximum value of R.P.U. belonging to the range of O.D. in exponential phase.

Materials

Long term glycerol stocks were stored at -80°C with a final glycerol concentration of 20%
Antibiotics were: Ampicillin (Amp) 100 ug/ml. All of them were stored at -20°C in 1000x stocks. Amp was dissolved in water.
LB medium was prepare with: 1% NaCl, 1% bactotryptone, 0.5% yeast extract. The medium was not buffered with NaOH.

User Reviews

UNIQcae3bbf981a0994e-partinfo-0000000E-QINU

•••••

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]
Promoter	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:

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:

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.

UNIQcae3bbf981a0994e-partinfo-0000004E-QINU

•••••

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.