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Part:BBa_J23101:Experience
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Applications of BBa_J23101
GreatBay_China 2018
Team GreatBay_China 2018 characterized J23119, Part:BBa_J23105, and Part:BBa_J23101 by assembling them with Part:BBa_B0034 and a sfGFPPart:BBa_I746916 on three vectors: pUC20 (copy number about 500/cell), pR6K (copy number about 15/cell), pSC101 (copy number about 2/cell). Then we measured the fluorescence by Flow Cytometry as a reference for the TALE stabilized promoter library.
The result indicate that the strength of J23119, J23105, and J23101 are about the same as described by team iGEM2006_Berkeley, and the fluorescence increases as the copy number of the vector increases
TU Eindhoven 2015
We participated in the InterLab Study and came to know this part as a strong promotor in comparison with J23106 and J23117. It inspired us to use this promotor also for the characterization of our mNeonGreen part [[1]].
Unexpected LuxR-AHL repressible behaviour
This promoter shows activity repression as a function of AHL in presence of LuxR [ADD REFERENCE LINK]. Results are shown below for a set of widely used promoters tested in the same conditions (TOP10, M9 medium supplemented with casamino acids and glycerol, assayed in a microplate reader). Promoters drive the BBa_I13507 RFP expression device. The TACTAGTG scar is present between promoter and RBS, except for PLlacO1 (BBa_R0011), PlacIQ (BBa_I14032) and PR (BBa_R0051) where the scar is TACTAGAG. RFP was measured and used to compute Scell (arbitrary units) or RPU values. Promoters were assembled in pSB4C5 and were co-transformed with BBa_S03119 in pSB3K3 in TOP10. Blue bars represent the activity when no AHL was added to the medium, while green bars represent the repressed activity (AHL 20 µM was added to the medium). Only BBa_J23101, BBa_J23102 and BBa_J23104 showed repression and, for them, percent repression is reported.
Kosuri 2013 Promoter-RBS composability paper
Kosuri et al investigated the composability of the promoters of the Anderson library and many others (114 total) with 111 popular RBS sequences, characterizing the relative strength of each component compared to all others by testing 12,563 combinations with DNAseq, RNAseq, and "FlowSeq". - MLC
Kosuri et al., [http://www.pnas.org/content/110/34/14024.long Composability of regulatory sequences controlling transcription and translation in Escherichia coli], Proc Natl Acad Sci U S A. 2013 Aug 20;110(34):14024-9. doi: [http://dx.doi.org/10.1073/pnas.1301301110 10.1073/pnas.1301301110]. Epub 2013 Aug 7.
Evaluation of Anderson promoter J23101 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_K823005 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 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 | |
| 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] |
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
UNIQf5b89b4c0284d2de-partinfo-0000000E-QINU
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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 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):
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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[http://2012.igem.org/Team:Uppsala_University Uppsala University 2012] |
iGEM Team Uppsala University 2012 Promoter strength 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.
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. |
UNIQf5b89b4c0284d2de-partinfo-00000051-QINU
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iGEM15_UNITN-Trento |
We used the part BBa_J23101 in the InterLab Measurement context. We assembled a measurement device by inserting the part BBa_I13504 amplified by PCR into the part BBa_J23101. The device was characterized in E. coli NEB10β, JM109, and NEB Express by measuring GFP expression with a cuvette-based spectrofluorometer, a fluorescence plate reader, and a flow cytometer. We also confirmed the promoter activity with a cell-free S30 extract system and measured mRNA by RT-qPCR. |
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.
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UNIQf5b89b4c0284d2de-partinfo-00000057-QINU
UNIQf5b89b4c0284d2de-partinfo-00000058-QINU
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UNIPV-Pavia iGEM 2011 |
NB: unless differently specified, all tests were performed in E. coli MGZ1 in M9 supplemented medium at 37°C in low copy plasmid pSB4C5.
Used to quantify promoter strength and the RBS efficiency.
BBa_J23101 is the reference standard promoter for the computation of RPUs. As discussed in 'data analysis' section, RPUs are relative units for the evaluation of promoter strength, based on a mathematical model of the transcription and the translation of a reporter gene. The RPUs are supposed to be indepedent on the experimental setup, provided that the reference standard BBa_J23101 must be assayed in the same experimental condition of the studied promoter. It means that if the studied promoter is in a low copy number plasmid and drives the expression of a reporter protein P, J23101 must be assembled in the same vector upstream of the same reporter P. This approach is in accordance with the philosophy of synthetic biology, based on the concept of 'modularity' of the components. According to this approach, the assembly of basic well characterized modules to build complex circuits allows the prediction of the circuit behavior starting from the knowledge on the basic parts. Salis et al. [Nat Biotec, 2009] stated that 'Identical ribosome binding site sequences in different genetic contexts can result in different protein expression levels' and again 'It is likely that this absence of modularity is caused by the formation of strong secondary structures between the RBS-containing RNA sequence and one protein coding sequence but not another.' For this reason, RPUs might not be reliable when comparing the same promoter with different RBSs because of the un-modularity of the RBS. In order to asses what's the effect of RBS 'un-modularity' on RPUs reliability, we have built a set of four constitutive promoters (BBa_J23101) followed by one of the four RBSs tested. These parts were used to evaluate RBS efficiency. Data were collected and analyzed as described in 'Measurements' and 'Data analysis' sections. RPUs and Synthesis rate per cell [AUr] were computed and results are summarized in the table below.
On the other hand, if the hypothesis of RBS modularity depending on the promoter is accepted, the J23101-RBSx series we have provided can be used as a library of ready-to-use reference standard for RPU evaluation, that allows to depurate RPU measurement from RBS effect, thus providing only the promoter strength.
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UNIQf5b89b4c0284d2de-partinfo-0000005B-QINU
Characterization
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iGEM2014 [http://2014.igem.org/Team:XMU-China# XMU-China] |
iGEM2014 [http://2014.igem.org/Team:XMU-China# XMU-China] has sequenced this device. The following data was taken from this device. |
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We choose the promoter J23101 as an in vivo reference standard for promoter activity. In order to get the different expression intensity of BioBrick backbones, we can connect the backbones with the same promoter and GFP generator. Finally we can use the intensity of fluorescent protein as a charaterization data to report the expression intensity of different backbones. Unfortunately, there are few data about it. So we detected the two different backbones as examples and hope the other teams can provide more relevant data. For devices BBa_I20260 and BBa_J23101 + BBa_E0240. Both devices consist of Anderson promoter J23101 and GFP generator BBa_E0240. When the device is constructed in backbone pSB3K3. A low copy number is in expectation, as a result, a weak fluorescence strength is shown. While the device is constructed in pSB1C3 which is a higher copy number vector, hence a stronger fluorescence strength, so that it can be obvious enough to be observed in naked eyes. So we reconstructed this part, and submit the improved part BBa_K1412716 and BBa_K1412924 to the parts registry. BBa_K1412716: BBa_I20260 (J23101-B0032-E0040-B0015), in the pSB3K3 vector.BBa_K1412924: BBa_J23101 + BBa_E0240 (B0032-E0040-B0015), in the pSB1C3 vector.Measurenment
Figure 1. The plot of optical density versus timeFrom the plot of optical density versus time, we can conclude that the growth rate of bacteria is become lower with time. We measured the samples three times parallelly, and we can know that the reproducibility of the data is acceptable. When we compare BBa_K1412716 with BBa_K1412924, we can get that their growth rate are almost equal.
Figure 2. The plot of RFUs versus timeFrom the plot of RFUs versus time, we can conclude that RFUs grows linearly with time. When we compare BBa_K1412716 with BBa_K1412924, we can get that the fluoresent expression intensity of BBa_K1412924 is higher than BBa_K141716, because the pSB1C3 backbone has a higher copy number, hence we can get a stronger fluorescence strength.
Figure 3. The plot of RFUs versus OD600From the plot of RFUs versus OD600, we can conclude that RFUs grow linearly with OD600 both BBa_K1412716 and BBa_K1412924. Because the fluoresent protein expression of each bateria is contain, so when the concentration of bacteria increase, the fluoresent expression increase too. When we compare BBa_K1412716 with BBa_K1412924, we can get that the fluoresent expression intensity of BBa_K1412716 is lower than BBa_K1412924.
Figure 4. The plot of RFUs/OD600 versus timeFrom the plot of RFUs/OD600 versus time, we know the RFUs/OD600 is a representation of the fluoresent expression intensity of unit bacteria. So we can get that the fluoresent expression intensity of BBa_K1412716 is lower than BBa_K1412924.
Protocol1. Transformed BBa_K1412924 into DH5α competent cells, coated plates, grown in incubator for 12 hrs at 37℃. 2. Inoculate a 5 ml cultures of supplemented LB medium and antibiotic (Chloromycetin or Kanamycin 50 μg/ml) with single colony from the plate. 3. Cultures were grown in conical flask for 16 hrs at 37℃ with shaking at 200 rpm in the table concentrator. 4. Cultures were diluted 1:100 into three 20 ml fresh LB medium and grown for 3 hrs at 37℃ with shaking at 200 rpm in the table concentrator. 5. Then transfered 650 μl of the culture to a 1.5 ml centrifuge tube, centrifuged and washed twice with phosphate-buffered saline ([http://en.wikipedia.org/wiki/Phosphate_buffered_saline PBS], pH 7.4) to minimize the background fluorescence from the medium. 6. The washed cells were suspended in [http://en.wikipedia.org/wiki/Phosphate_buffered_saline PBS] and diluted to bring the cells into an appropriate concentration range (2–5 times) before taking fluorimeter measurements. 7. Measure the fluorescence and absorbance: (1)Fluorescence:
(2)OD600 (optical density at 600 nm):
8. Measure every 30 minutes in the next 4 hrs. References[1] [http://journals.aps.org/pre/abstract/10.1103/PhysRevE.82.021911 Bagh, Sangram, Mahuya Mandal, and David R. McMillen. "Minimal genetic device with multiple tunable functions." Physical Review E 82.2 (2010): 021911] More information, click here: [http://2014.igem.org/Team:XMU-China# XMU-China]</i> |
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<I>[http://2015.igem.org/Team:Uppsala iGEM Uppsala 2015] |
iGEM Uppsala 2015 reviewThe results below is from Uppsala iGEM team 2015 The constitutive promoters J23101 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. 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 |
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[http://2019.igem.org/Team:Tacoma_RAINmakers Tacoma RAINmakers 2019] |
Tacoma RAINmakers 2019: Relative Strength of Anderson Promoters
We collected characterization data on the relative RFP expression rates of Anderson Promoters BBa_J23100, BBa_J23101, BBa_J23102, BBa_J23105, BBa_J23106, and BBa_J23112 in the RFP expression plasmid BBa_J61002 (samples provided in the 2019 iGEM Distribution Kit). 
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UNIQf5b89b4c0284d2de-partinfo-0000007A-QINU
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University of Texas at Austin iGEM 2019 |
UT Austin iGEM 2019: Characterization of metabolic burden of the Anderson SeriesDescriptionThe 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. This GFP expression rate fluctuates depending on the number of ribosomes available for the host cell to sustain itself. Using this strain, the relative burden (percent reduction in growth rate) of each Anderson Series part was determined. 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]
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. </table> |
Characterization in a cell free system - BOKU-Vienna 2020
The main goal of this series of experiments was to compare the expression strength of 3 constitutive promoters when used in a cell free expression system.1 This expression system includes the core RNA polymerase and sigma 70 transcription factor. Three closely related promoters of well documented expression strength controlled by sigma 70, as well as a single terminator (T_B1001) were chosen, as described in the parts section. The promotores are: BBa_J23101 for better readability called “101”, BBa_J23105 “105” and BBa_J23109 “109”. To accurately measure expression strength the fluorescent protein mCherry was utilized as the expressed protein, as one can deduct expression strength based on fluorescence measurements. The complex of promotor, gene and terminator was then assembled via Golden Gate cloning into the Golden Gate backbone 02, which additionally contains an Ampicillin resistance gene as a selection marker:
| Reaction Mix | Master Mix | |
|---|---|---|
| x1 | x5 | |
| [μL] | [μL] | |
| BB2_AB_14 [40 nM] | 1 | 5 |
| BB1_Prom (101/105/109) [40 nM] | 1 | - |
| BB1-mCherry [40 nM] | 1 | 5 |
| BB1-Term [40 nM] | 1 | 5 |
| Cutsmart buffer2 | 2 | 10 |
| ATP | 2 | 10 |
| BbsI - HF [20 U/µL] | 2 | 10 |
| T4 ligase [120 U/µL] | 0.25 | 1.25 |
| H2O | 9.75 | 48.25 |
| total volume | 20 | 95 |
With this assembled plasmid, a transformation was carried out. The transformation was then distributed on Agar plates lazed with Ampicillin as a selection marker. From these plates colonies were picked to prepare liquid overnight cultures, from which the DNA was purified using Miniprep kits.3 The success of the Golden Gate Cloning was then confirmed utilizing restriction digests as well as outsourced sequencing.
Experimental setupDue to the small volume of cell free system expression reactions (~20 µL), it would not have been feasible to take samples over time comparable to a fermentation process. Instead several reactions were set up at different time intervals in order to get an as accurate as possible picture of the fluorescence development over the timeframe in question. To increase the validity of the experiment, doublets were prepared. The reactions consisted of the following components:
| Table 1. Components of assay 1 | Volume [µL] |
|---|---|
| myTXTL® Sigma 70 Master Mix | 9 |
| Plasmid DNA [10,8 nM] | 9 |
| Table 2. Components of assay 2 | Volume [µL] |
|---|---|
| myTXTL® Sigma 70 Master Mix | 15 |
| Plasmid DNA [20 nM] | 5 |
Results
The measured fluorescence is presented in fluorescence units [FU].
Table 3. Fluorescence of mCherry at assay 1:
| Time [h] | 101[FU] | 105[FU] | 109[FU] |
|---|---|---|---|
| 1.3 | 8.0 | 6.5 | 6.0 |
| 2.8 | 7.5 | 6.5 | 6.0 |
| 4.8 | 28.0 | 6.5 | 6.0 |
| 8.0 | 57.0 | 14.0 | 7.0 |
The data clearly shows that the respective expression strength in E. coli (101>105>109) remains the same in this cell free expression system.4 However, the difference between 101 compared to 105 and 109 is far more pronounced than the difference between 105 and 109. To see the differences better, especially between 105 and 109, a second experiment was carried out. For this, plasmid DNA with higher concentration was produced. Through this, a higher concentration of cell free reaction mix was possible. The different parameters can be seen in table 2 compared to table 1.
Table 4. Fluorescence of mCherry at assay 2:
| Time [h] | 101[FU] | 105[FU] | 109[FU] |
|---|---|---|---|
| 3.45 | 534 | 7.5 | 6 |
| 5.03 | 937 | 9.5 | 5.5 |
| 7.45 | 1310.5 | 18.5 | 4.5 |
| 9.45 | 1082 | 19 | 5.5 |
Interestingly, a drop-off in fluorescence past 7.5 hours can be seen for 101. This might be the result of experimental errors and different operators. This seems as a more coherent explanation than a decay of protein. Even though the incubation time was longer and the concentration of the cell free expression system higher, the difference between 105 and 109 is hardly visible. In fact, 109 showed no activity at all as can be seen in table 4.
Conclusion
To produce a notable amount of a protein of interest, the promoter 101 seems to be the best choice. 105 shows some activity compared to 109 which produced no measurable amount of protein. This result seems valid. However, this result is meaningful for this very expression system.
1myTXTL® Sigma 70 Master Mix Kit
250 mM Potassium Acetate, 20 mM Tris-acetate, 10 mM Magnesium Acetate, 100 µg/ml BSA
3Monarch Plasmid Miniprep Kit
4https://parts.igem.org/wiki/index.php?title=Part:BBa_J23101