Part:BBa_R0010:Experience

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For assay, GFP generator BBa_E0240 was placed under the control of BBa_R0010.

Promoter Activity in Varying Concentrations of Glucose and IPTG

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 R0010.

Figure 1. The distribution of the sfGFP expression tested by flow cytometry with promoter R0010 and Csy4 insulator.

Figure 2. The intensity of different promoters is shown by the column height and the randomness of their functions by the bar width.

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••••• Antiquity

This review comes from the old result system and indicates that this part worked in some test.

••• UNIPV-Pavia iGEM 2010

BBa_R0011 hybrid lac promoter and the BBa_R0010 wild type lac promoter were characterized at different copy number in TOP10 E. coli strain. This strain contains a lacI expression system in the genome. Induction static transfer function (computed in Relative Promoter Units), dynamics and metabolic burden were evaluated as a function of different IPTG concentrations in M9 supplemented with glycerol growth medium. A RFP generator (BBa_I13507) was used as a reporter gene. In particular, these measurement systems were used: pSB1A2-BBa_J107010 pSB1A3-BBa_J04450 pSB4C5-BBa_J107010 pSB4C5-BBa_J04450 At first, BBa_J107010 and BBa_J04450 inducibility was tested in a high copy number vector (pSB1A2 or pSB1A3). The results are shown here as the relative RFP synthesis rate per cell. Relative RFP synthesis rate per cell in pSB1A2-BBa_J107010 and pSB1A3-BBa_J04450. The error bars represent the standard errors of three independent measurements. Results show that in this condition BBa_R0010 is about 2-fold stronger than BBa_R0011, but induced and uninduced cultures did not show differences in the RFP signal. This result is expected because the vectors are propagated at about 200 copies per cell, while the lacI repressor is present at single copy in the genome and thus it is not able to repress the lac promoters in such high copy. The doubling times and their standard errors estimated from data are reported below for pSB1A2-BBa_J107010 and pSB1A3-BBa_J04450 with and without 1mM of IPTG. Cultures Mean doubling times [minutes] standard errors over 3 independent experiment [minutes] pSB1A2-BBa_J107010 77,7 3,1 pSB1A2-BBa_J107010 + 1mM IPTG 76,5 2,2 pSB1A3-BBa_J04450 107,8 0,3 pSB1A3-BBa_J04450 + 1mM IPTG 101,7 4,8 These results demonstrate that cells growth is not significantly affected by the presence of IPTG, even at the high of 1 mM. BBa_J107010 and BBa_J04450 were then tested in the low copy (~5 copies per cell) vector pSB4C5 in order to test their inducibility. The results are shown here as the RPU values at the steady state (constant RFP sysnthesis rate per cell) at different IPTG concentrations. RPU of pSB4C5-BBa_J107010 and pSB4C5-BBa_J04450 as a function of IPTG concentration. The error bars represent the standard errors of three independent measurements. Results show that in this condition both BBa_R0010 and BBa_R0011 produce different amounts of RFP as a function of the IPTG concentration. The amplitude of the two curves show that the promoters are very strong when induced with IPTG >= 10 uM. Although the experiments were carried out in the same conditions, the variability between experiments was high, especially for BBa_R0010 (mean coefficient of variaton of about 37% for BBa_R0010 and 15% for BBa_R0011), while the RPU variability between three wells in the same experiment is much lower (mean coefficient of variaton of bout 3.5% for both promoters). The above figure shows that BBa_R0011 is stronger than the BBa_R0010 wild type promoter in low copy plasmid. This result is unexpected because the same promoters in high copy vectors behaved differently (BBa_R0010 was stronger than the BBa_R0011, see above). In the uninduced state, BBa_R0011 has about the same strength as the BBa_J23101 reference standard promoter. This static characteristic shows that the promoters are both leaky and a very low IPTG concentration (10 uM) is sufficient to trigger gene expression at *very* high levels. These results demonstrate that the genomic lacI is partially able to repress the two promoters, but very low IPTG concentrations are sufficient to bind the repressor and trigger the promoters transcription. Doubling times were also estimated for these cultures. Their values are reported below for uninduced and 1 mM IPTG-induced cultures. Cultures Mean doubling times [minutes] standard errors over 3 independent experiments [minutes] pSB4C5-BBa_J107010 113,5 10,8 pSB4C5-BBa_J107010 + 1mM IPTG 106,8 5,5 pSB4C5-BBa_J04450 85 5 pSB4C5-BBa_J04450 + 1mM IPTG 90 4,5 As obtained for the cultures with high copy plasmids, the growth rate of TOP10 harbouring low copy vectors with the measurement parts is not affected by IPTG presence. Dynamic characterization in low copy vector: The figure below shows a typical relative RFP synthesis rate per cell time series for BBa_J107010 and BBa_J04450 induced with 1 mM of IPTG and uninduced. These time series show that the full induction can be reached after about 50 min from the induction. Mean Scell signal as a function of time for pSB4C5-BBa_J107010 and pSB4C5-BBa_J04450. Induced (with 1 mM of IPTG) and uninduced cultures are shown. Induction occurs at t=0. The shown graph is relative to one of the three experiments performed in different days. Conclusion: the characterization of two IPTG-inducible promoters has been performed and the performance of these two promoters have been compared in terms of transcriptional strength. The reported results are easily sharable in different laboratories thanks to the used standard RPU approach. Methods: A of long term storage glycerol stock was streaked on a LB plate with suitable antibiotic. Tha plate was incubated overnight at 37°C. A single colony was inoculated in 1 ml of M9 + suitable antibiotic in a 15 ml tube and incubated at 37°C, 220 rpm for about 16 hours. The grown cultures were then diluted 1:100 in 2-5 ml of M9 supplemented medium and incubated in the same conditions as before for about 4-5 hours. For each desired IPTG concentration to be tested, three 200 ul aliquots of the cultures 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). 2 ul of properly diluted IPTG (Sigma Aldrich) were added to the three wells for each desired concentration. 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 80; O.D. filter at 600 nm; RFP filters at 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. This experiment was performed three times in different days. Data analysis: Relative Promoter Units (RPUs) were estimated as described by [Kelly JR et al. (2009), J Biol Eng 3:4]. Briefly: Absorbance and fluorescence time series were normalized by subtracting the absorbance of the media and the fluorescence of a negative control (a non fluorescent TOP10 culture) respectively, thus yielding O.D.600 and RFP time series. RFP synthesis rate per cell (called Scell) was computed as (1/O.D.600)*dGFP/dt. (this signal is not actually the RFP synthesis rate, but is proportional to it). The RFP synthesis rate per cell was averaged at the steady state during the exponential growth phase (validated by identifying the linear region of the ln(O.D.600)). The RPU of the promoter of interest in a specific condition was computed as mean_Scell,phi/mean_Scell,J23101 where phi is the promoter of interest, J23101 is the reference standard and mean_Scell is the mean Scell signal value, computed as explained above.

••••• iGEM Copenhagen

We attempted to use this BioBrick to assembly a protein expressing device. We were unable to do it with standard assembly but had succesful result utilizing the 2011 [http://2011.igem.org/Team:Copenhagen/Collaboration| DTU-2 user assembly plug'n'play method]

••••• iGEM Amsterdam 2011

This promoter is always active in Top 10 E. coli, because it lacks the LacI protein. Our findings seem to confirm this when used in combination with CspC.

••••• iGEM at UC Davis 2011

Our team used BBa_R0010 extensively in the generation of a LacI promoter mutant library. Our goal was to thoroughly characterize both the LacI wild type, R0010, and our seven variants (listed in the registry as parts BBa_K611021-BBa_K611027). To characterize these parts, we used our LacI promoter characterization plasmid, BBa_K611013. The LacI characterization plasmid (K611013): This plasmid allowed us to ligate in any promoter upstream of the first RBS and have it drive the expression of GFP. The later half of the part is comprised of the pBad promoter in front of the coding region for the LacI repressor (BBa_C0012). In the presence of AraC, the pBad promoter is inducible by arabinose. Therefore, when using the LacI characterization plasmid, one can increase the concentration of repressor by adding arabinose to the system. Similarly, LacI can be derepressed by IPTG. Our goal was to characterize R0010 and our seven mutants at several levels of both repressor concentrations induced by arabinose and IPTG concentration. The following figures display our measurements for relative fluorescence to the wild type of each promoter. ••••• iGEM OLS_Canmore 2016 We attempted to use this part to express keratinase genes (BBa_K1717171 and BBa_K1717172) in E.coli. This was unsuccessful but not because of the function of the promoter, rather because of the low level of secretion of keratinase in E.coli. The above plot shows the relative fluorescence of BBa_R0010 in the LacI characterization construct (BBa_K611013) for the various repressor and IPTG levels at which we tested. The plot is normalized to its maximal value, such that we could measure the mutants relevant to it. For arabinose induction of the repressor, we tested from a range of 0 percent by weight to 0.0014 percent by weight, which we believe best represents minimal repressor level to near maximal repressor level. After much testing, we also concluded that the range from 0 mM to 5 mM IPTG best demonstrated the various levels of LacI induction. These measurements were acquired using a fluorescence plate reader. The typical set up of each run involved using a 96 well plate with each well containing 135 uL of LB, 15 uL of the proper IPTG concentration, and the inoculated culture in the BW22826 strain. Each point was obtained through testing in triplicate, allowing us to do seven arabinose levels, four IPTG levels, and controls of wild type, LB, and DH5α on each 96 well plate. After characterizing the BBa_R0010 wild type, we characterized our 7 mutant promoters using the same methods. With these R0010 mutants, we have significantly widened the functionality of this part. Promoters are integral to the function of all genetic circuits, thus it is especially important that they be available in a broad selection and that their activity is well-documented and predictable. Each variant is different and useful in its own way, with altered characteristics such as promoter strength or repressor binding affinity, as can be seen in this graph and in the 3-dimensional plotting widget on our wiki. R0010 is a very important part, and its ability to be repressed opens up a huge range of possible circuits. This mutant library is very useful for any user that wants to incorporate a LacI repressible promoter into their circuit, but would like to use a promoter with different levels of activity. Our hope is that this mutant library will promote the use of BBa_R0010 and encourage other contributors to the registry to make mutant libraries of their own.

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••••• Calgary 2012

This part was used in constructing electrochemical reporter test circuits. Using the uidA gene as a reporter, accurate real-time data was obtained for the expression of induced and uninduced R0010 in TOP10 E. coli. The uidA gene cleaves the chemical para-nitrophenol-β-D-glucuronide (PNPG) into para-nitrophenol (PNP), which can be detected at an electrode held at 1.6V vs the Reduction of Hydrogen Electrode (RHE). 3mL of an overnight culture was induced with 100µM IPTG at time zero in a 25mL 0.1M pH7 PBS solution and the production of PNP was measured. The expression patterns are shown below. For more info see our wiki. Potentiostatic detection of PNP at 1.6V vs RHE by either an IPTG induced uidA gene or leaky expression of an uninduced uidA gene.

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••••• [http://2012.igem.org/Team:Uppsala_University 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. LacI repression The possibility of repression by lacI and induction by IPTG was evaluated in the pSB4C15Iq backbone. Cells grown with IPTG (0.5 mM) had a 100-fold increase of RFP expression, when compared to those grown without. Read about pSB4C15Iq for details.

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Review must be between 0 and 5 iGEM2016_ Hong_Kong_HKUST

The project of our team this year is to build a Tristable switch. Our design aims to achieve three main characteristics: stable and distinct signal output, and fast state-switching rate. In which stability is the major goal that we would like to achieve. As a results, the strength of the promoters need to be similar. As a result, we compared the promoter strength of phlFp, tetp and lacp by ligating them with a GFP reporter gene, BBa_E0240, in BioBrick RFC10 standard. Results Experiments on comparing the strength of phlFp, tetp and lacp, were performed. Results indicated that phlFp is the strongest among the three, with 1.96 times and 6 times stronger than that of tetp and lacp, respectively. Fig 2. Comparison on the strength of phlFp, tetp and lacp. Negative control represents BBa_E0240. Characterization was done using E. coli strain JW0336. Cells were first precultured overnight and were subcultured to mid-log phase where GFP emission measurements were made using an EnVision® multilabel reader. This result was obtained by combining 3 characterization data obtained in 3 different days. Error bar present SD from 3 biological replicates.

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The induction of R0010 by lactose in M9 medium was measured by 2022 HS_China iGEM team

We overnight cultured engineered bacteria containing plasmid BBa_K4183005 in 5 ml LB medium and subsequently inoculated them into medium containing M9 (with different carbon sources or IPTG) and incubated at 37°C in a fully automated ELISA, with plate shaking every 15 min and data measured every 30 min.

The results showed that EcN growth was consistent under different carbon sources (glucose/lactose) (Figure 1A), and all of them could activate the expression of R0010. The expression intensity of R0010 was higher when the carbon source was D-glucose (Figure 1B). However, when the carbon source was β-lactose, the expression intensity of R0010 was low with or without the addition of IPTG (Figure 1B).

Figure 1. Figure 1. Growth of EcN and expression of R0010 in M9 medium under different conditions. (A) OD600 of each well was measured at 0.5 h intervals in M9 medium under different conditions. (B) Fluorescence intensity of each well was measured at 0.5 h intervals in M9 medium under different conditions with excitation light at 485 nm and emission light at 510 nm, N=3.

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