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Here we describe the characterization results of 4 parts of our own design, 2 existing parts re-built because they were inconsistent and 7 existing parts taken from the Registry. When not reported differently, all the experiments have been performed according to Growth conditions and Data analysis sections.

Our new parts:

• BBa_K173003 - ethanol producing device
• BBa_K173007 - aTc inducible device with J23100 promoter
• BBa_K173011 - aTc inducible device with J23118 promoter
• BBa_K173010 - lactose/IPTG inducible device with J23118 promoter

Re-built existing parts (BBa_our part code/BBa_existing part code):

• BBa_K173004/BBa_I732019 - beta-galactosidase protein generator
• BBa_K173005/BBa_Q04400 - tetR QPI

Existing parts from the Registry:

• BBa_J23100, BBa_J23101, BBa_J23118 - constitutive promoter family members
• BBa_F2620 - 3OC6HSL receiver device
• BBa_K116001 - nhaA promoter
• BBa_K112808 - Enterobacteria phage T4 Lysis Device
• BBa_R0011 - Plac hybrid promoter

Our new parts

BBa_K173003 - ethanol producing device

Description

This device takes PoPS as input and produces pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adhB) enzymes.

Pyruvate decarboxylase (pdc, ) catalyses the decarboxylation of pyruvic acid to acetaldehyde and carbon dioxide, while alcohol dehydrogenase II (adhB, ) catalyses the acetaldehyde reduction to ethanol. The latter enzyme, as reported in literature, is also able to work in the opposite direction, oxidizing ethanol to acetaldehyde. The two enzymes come from Zymomonas mobilis ethanologenic bacterium and constitute an essential step of alcoholic fermentation.

So, this device contains the minimum set of genes that are required to engineer a heterologous fermentation pathway.

The coding sequences of pdc and adhB genes have been optimized for Escherichia coli codon usage.

Characterization

Qualitative phenotype characterization

PLATES RESULTS

This device has been cloned downstream of 4 different promoters, one of which in two different vectors. These assemblies were transformed in TOP10 and gave the following phenotypes on LB agar plates and also on LB agar plates + 2% glucose (all the plates have been incubated at 37°C for about 11 hours, then some colonies were picked with a sterile tip and plates were incubated for additional 10 hours):

These results demonstrate that a constitutive expression of pdc and adhB gives a high metabolic burden to E. coli, in fact the only normal phenotype on plates was with non-induced device. This assembled part has been submitted to the Registry as .

BBa_F2620-BBa_K173003 in pSB1AK3 plate: example of normal size colonies.

BBa_K173005-BBa_K173003 in pSB1AK3 plate: example of small size colonies.

LIQUID CULTURES RESULTS

Grown cultures of and after the FIRST INOCULUM showed different phenotypes (qualitative analysis), as a function of the used protocol.

PROTOCOL#1 RESULTS (i.e. shaken anaerobic falcon tubes, no glucose):

PROTOCOL#2 RESULTS (i.e. unshaken anaerobic falcon tubes, 2% glucose):

PROTOCOL#3 RESULTS (i.e. shaken anaerobic falcon tubes, 2% glucose):

These results confirm the metabolic burden given by this BioBrick when gene expression is triggered: induced strains bearing a high copy plasmid containing do not survive, while low copy ones can survive even at high induction (1 uM). We decided to exploit the lux promoter leakage activity for in pSB1AK3 (high copy), while we decided to induce in pSB4C5 (low copy) the with 1 uM of 3OC6-HSL during our fermentation experiments.

The qualitative phenotypes of the grown cultures of and after fermentation in 10% glucose showed that pellets were higher in (in both pSB1AK3 uninduced and pSB4C5 induced) than in and in the negative control .

Moreover, after both inoculum steps, the grown cultures of and (in both pSB1AK3 uninduced and pSB4C5 induced) showed a "sparkling" phenotype, while and in the negative control did not.

Quantitative characterization

pH

pH has been measured in grown cultures after the FIRST INOCULUM (in 2% glucose after 24 hours) and after the SECOND INOCULUM (in 10% glucose after 24 or 48 hours). The measurements performed are the following:

PROTOCOL#1: pH at the end of fermentation (after the SECOND INOCULUM).

PROTOCOL#2: pH after the FIRST and the SECOND (end of fermentation) INOCULUM

PROTOCOL#3: pH after the FIRST and the SECOND (end of fermentation) INOCULUM

The shown results suggest that in strains bearing expressed pdc and adhB the fermentation does not give high levels of organic acids as in negative control strains which do not have pdc and adhB. It is surprising that the pH of in pSB1AK3 (pdc and adhB without any promoter) is slightly higher than in the negative controls. This could be due to a weak spurious transcription of pdc and adhB, amplified by the high copy number plasmid. This may re-direct part of pyruvate metabolism to ethanol and not to organic acids. Of course this phenomenon should be further investigated.

Growth curves

and its suitable negative controls growth was tested in our microplate reader: 200 ul aliquots (3 or 4 replicates) were taken from a just inoculated 30 ml culture (LB + 10% glucose) of the SECOND INOCULUM of  PROTOCOL#3 and let grow in the microplate reader according to the automatic protocol described in  Microplate reader experiments. The microplate was "sealed" with parafilm in order to create an anaerobic condition.

Growth curves of , in pSB1AK3, in pSB4C5 + 1uM HSL, in pSB1A2 negative control, in pSB1AK3 negative control

This test showed that in pSB1AK3 is able to reach much higher OD600 than the other cultures, reaching ? values. in pSB4C5 both induced and uninduced had comparable growth curves and reached OD600 of about ?. Negative controls ( in pSB1A2 and in pSB1AK3) had comparable growth curves and reached low OD (about ?). It is surprising that in pSB1AK3 reached a higher OD (?) than the negative controls.

The shown results are in complete accordance with the pH results, confirming that strains bearing in pSB1AK3 and in pSB4C5 produce less organic acids than the negative controls, hopefully thanks to pdc and adhB and consequently grow better.

Ethanol production Here we report the fermentation results of 100 g/l of glucose:

PROTOCOL#1: ethanol at the end of fermentation.

PROTOCOL#2: ethanol at the end of fermentation.

PROTOCOL#2: ethanol at the end of fermentation.

PROTOCOL#3: ethanol at the end of fermentation.

Conclusions

BBa_K173007 - aTc inducible device with J23100 promoter

Description

This is an aTc sensing device.

promoter drives the constitutive production of tetR repressor (), which inhibits tetR promoter () activity. When aTc is added to the medium, it binds tetR and inhibits it. So, the PoPS output is a function of the aTc concentration.

A tight regulation is expected for this inducible system because BBa_J23100 is a strong promoter and so tetR repressor should be produced at extremely high levels.

The data below are referred to , which is the measurement system of .

Characterization

Conclusions

We demonstrated that this part works as expected, sensing the aTc concentration provided in the culture medium. The transfer function of this sensor has been characterized in standard units (RPUs) in two different growth media (LB and M9 supplemented with glycerol), as well as the metabolic burden (in terms of doubling time) which affects E. coli bearing this part.

On the other hand, we did not expect to have a higher GFP synthesis rate per cell after the exponential growth phase than in the exponential phase itself (as reported in the 3rd plot).

COMMENTARE QUI IL TIGHT REGULATION

BBa_K173011 - aTc inducible device with J23118 promoter

Description

This is an aTc sensing device.

promoter drives the constitutive production of tetR repressor (), which inhibits tetR promoter () activity. When aTc is added to the medium, it binds tetR and inhibits it. So, the PoPS output is a function of the aTc concentration.

A less tight regulation is expected for this inducible system than in because BBa_J23118 promoter is weaker than BBa_J23100 and so tetR repressor shold be produced at lower levels than in the other sensor.

The data below are referred to , which is the measurement system of .

Characterization

Conclusions

We demonstrated that this part works as expected because GFP is produced as an increasing function of the aTc concentration provided in the culture medium. The transfer function of this sensor has been characterized in standard units (RPUs) in two different growth media (LB and M9 supplemented with glycerol), as well as the metabolic burden (in terms of doubling time) which affects E. coli bearing this part.

On the other hand, as for BBa_K173007, we did not expect to have a higher GFP synthesis rate per cell after the exponential growth phase than in the exponential phase itself (as reported in the 3rd plot).

COMMENTARE QUI LA PARTE DEL LEAKAGE E CONFRONTARLA CON K173007

BBa_K173010 - lactose/IPTG inducible device with J23118 promoter

Description

This should work as a lactose/IPTG sensor.

promoter drives the constitutive production of lacI repressor (), which inhibits lac promoter () activity. When lactose or IPTG is added to the medium, it binds lacI and inhibits it. So, the PoPS output is a function of lactose/IPTG concentration.

Thanks to the hybrid lac promoter (), designed taking the Plambda promoter () and substituting its cI () binding sites with two lacI binding sites, the behaviour of this device is not a function of glucose concentration because the wild type CAP binding sites are not present in this artificial lac promoter.

The test performed to characterize this device has been done with , which is the measurement system of itself.

Characterization

Conclusions

We did not perform any standard measurement on this device because preliminary tests showed that GFP levels of induced and non induced cultures were the same and were equals to negative control.

Unfortunately, we did not check if the sequence of , so we do not know if it was actually correct. Another possibility is that lacI is produced at so high levels that 2 mM of IPTG is not sufficient to induce the lac promoter.

Further tests should be done for this system.

Re-built existing parts

BBa_K173004/BBa_I732019 - beta-galactosidase protein generator

Description

This is a beta-galactosidase protein generator with strong RBS.

We built up . It is a twin of , which was classified as "inconsistent" by iGEM HQ in 2008 and so we decided to improve this part submitting a new consistent DNA to the Registry.

This part takes PoPS as input to express lacZ gene (), encoding for beta-galactosidase enzyme. This enzyme can be used to cleave lactose molecule to glucose and galactose, but can also be used as a reporter protein for colorimetric assays (together with X-Gal or ONPG as a substrate).

X-gal is cleaved by β-galactosidase yielding galactose and 5-bromo-4-chloro-3-hydroxyindole. The latter is then oxidized into 5,5'-dibromo-4,4'-dichloro-indigo, an insoluble blue product.

Characterization

Conclusions

We improved the existing part building up and testing its activity. This new part has been submitted to the Registry, as well as its physical DNA, allowing future users to assemble this beta-gal protein generator in their own project.

This part has shown to work as expected when a PoPS input is given, being able to cleave X-Gal on LB agar plates. In our case, we have tested this part with upstream, which provides a promoter strength of ? RPU in LB medium.

Further comparative tests should be done in order to see if lactose cleavage can be performed faster than in wild type E. coli, after the choice of a suitable promoter which controls this protein generator.

BBa_K173005/BBa_Q04400 - tetR QPI

Description

Characterization

Conclusions

Existing parts from the Registry

BBa_J23100, BBa_J23101, BBa_J23118 - constitutive promoter family members

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 , and that are the measurement parts of respectively , and .

Characterization

Conclusions

RPU estimation of these promoters was not present in the Registry and even the doubling time of these parts was not documented. We added these data in the pages of , and characterized parts, hoping that they can be useful for promoter comparison in standard units.

If we consider the promoter ranking, provided in saturation phase in the [http://partsregistry.org/Promoters/Catalog/Anderson Anderson Promoter Collection Registry page], the estimated strength in RPU of BBa_J23100 and BBa_J23118 are in accordance with these values: INSERIRE I VALORI E COMMENTARE

Note: plasmid is equals to pSB1A2 with a RFP expression system downstream of the cloning site.

BBa_F2620 - 3OC6HSL receiver device

Description

This device gives PoPS as output and can be induced with 3OC6-HSL autoinducer molecule: it binds luxR protein (encoded by ), which is constitutively expressed by tetR promoter (). LuxR-HSL complex can work as a transcriptional activator for lux promoter ().

Several studies have been performed on this BioBrick. Here we provide the experimental characterization we performed during this summer. The tests have been performed through measurement system, which has a GFP protein generator downstream.

Characterization

Conclusions

The induction curve of the receiver device, reported in page (M9 supplemented medium), was represented in PoPS units, while ours is reported in RPUs and has been obtained through a very similar protocol (see Growth conditions section). Anyway, the experiments we performed in M9 supplemented medium confirmed the induction curve shape of this device, with a switch point between ? and ?.

We also estimated this transfer function of this device in LB medium, for which no data were reported in the Registry.

BBa_K116001 - nhaA promoter

BBa_K116001 from iGEM 2008 NYMU-Taipei

We received this BioBrick from iGEM in September but the bacterial strain that contained the plasmid wasn't declared. So we decided to sequence it as check and transform it into E.coli TOP10.

We wanted to perform some experiments to better understand how it works and if can be successfully used.

We performed several experiments with different LB medium and we got almost the same results. We used:

• LBK (NaCl 0M) (pH 5.5 - 6.6 - 7.5 - 8.5)
• LB NaCl 70mM (pH 5.5 - 6.6 - 7.5 - 8.5)
• LB NaCl 171mM (pH 5.5 - 6.6 - 7.5 - 8.5)
• LB NaCl 250mM (pH 5.5 - 6.6 - 7.5 - 8.5)
• LB NaCl 600mM (pH 10 - 11.2)

Here we show just two experiments to explicate our work. You can download the complete list from this link.

Experiment Na+ 0M

Motivation

In our opinion the working principle of the antiporter Na+/H+ channel described in [Rachel Karpel et al., Etana Padan et al., N. Dover et al.] makes the nhaA promoter a Na+ sensor and only under certain conditions (presence of Na+) a pH sensor.

Methods

• We prepared falcons of LBK (potassium - 87mM - instead of sodium) and adjusted pH using KOH and HCl to values 5.5, 6.6, 7.5 and 8.5.
• We inoculated 8ul of Invitrogen TOP10 containing BBa_K116002 into 4ml of LB + Amp and incubated overnight at 37°C, 220 rpm. We did the same for TOP10 with BBa_K173001 and BBa_B0033 inside.
• Next morning we put 50ul from each of the three falcon into 5ml of LBK pH 6.6 and incubated again for about four hours and a half at 37°C, 220 rpm.
• We measured the final OD with TECAN F200 and diluted each genetic circuit into four falcons with LBK at different pH (5.5 - 6.6 - 7.5 - 8.5) in order to obtain a same OD equal to 0,02 (12 falcons overall).
• Then we performed a 6 hours' experiment with measures of absorbance and fluorescence every 5 minutes with TECAN F200. Each value shown is the mean of three measures.

Results

BBa_K116001 Absorbance	BBa_K116001 Fluorescence	pH 5,5: Fluorescence
pH 6,6: Fluorescence	pH 7,5: Fluorescence	pH 8,5: Fluorescence

Comments

As expected BBa_K116001 didn't produce any GFP. So we can consider it a Na+ sensor and only secondarily a pH sensor.

Experiment Na+ 250mM

Motivation

We’ll try again to make E.coli producing GFP at the variation of pH.

Methods

• We prepared falcons of LB NaCl 250mM and adjusted pH using KOH and HCl to values 5.5, 6.6, 7.5 and 8.5.
• We inoculated 8ul of Invitrogen TOP10 containing BBa_K116001 into 4ml of LB + Amp and incubated overnight at 37°C, 220 rpm. We did the same for TOP10 with BBa_K173001 and BBa_B0033 inside.
• Next morning we put 50ul from each of the three falcon into 5ml of LB NaCl 250 mM pH 6.6 and incubated again for five hours and at 37°C, 220 rpm.
• We measured the final OD with TECAN F200 and diluted each genetic circuit into four falcons with LB NaCl 250mM at different pH (5.5 - 6.6 - 7.5 - 8.5) in order to obtain a same OD equal to 0,02 (12 falcons overall).
• Then we performed an experiment of 21 hours duration with measures of absorbance and fluorescence every 5 minutes with TECAN F200. Each value is the mean of three measures.

Results

BBa_K116001 Absorbance	BBa_K116001 Fluorescence	pH 5,5: Fluorescence
pH 6,6: Fluorescence	pH 7,5: Fluorescence	pH 8,5: Fluorescence

Comments

We didn't expect this. After looking better for a motivation in some articles ([Rachel Karpel et al.]) we think this could be because of the E.coli strain: we use TOP10 while a special strain (delta-pump) without some membrane proteins that regulate E.coli homeostasis is used in other experiments.

Final considerations

In our opinion this sensor (primarily sodium sensor and secondarily pH sensor) needs very particular conditions to work (first of all a specific bacterial strain) we couldn’t reproduce, so we consider it almost unusable.

BBa_K112808 - Enterobacteria phage T4 Lysis Device

Description

Characterization

Clonclusions

BBa_R0011 - Plac hybrid promoter

Description

The hybrid lac promoter (BBa_R0011) has been designed taking the Plambda promoter (BBa_R0051) and substituting its cI (BBa_C0051) binding sites with two lacI binding sites.

This promoter can be repressed by lacI (BBa_C0012), which can be repressed by lactose or IPTG, providing a lactose/IPTG inducible system. Differently from wild type lac promoter, this part does not have any CAP binding sites, so its behaviour is glucose-independent.

Even if lacI is not expressed in this BioBrick, strains bearing a genomic copy of lacI can repress this promoter, which acts as a glucose-independent lactose/IPTG sensor. In the other strains BBa_R0011 acts as a constitutive promoter.

Here we provide the characterization of this promoter in E. coli TOP10, which has a lacI genomic copy, constitutively expressed in a weak manner.

The data below are referred to , which is the measurement system of .

Characterization

Conclusions

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 OD600 of 0.02 (measured with a microplate reader, 200 ul of volume per well, not with a 1 cm pathlength cuvette) in a sufficient amount of medium to fill all the desired microplate wells.
• These new dilutions were aliquoted in the microplate, avoiding to perform dynamic experiments in the microplate frame (see Measurement 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;
  • sample time of 5 minutes;
  • fluorescence gain of 50;
  • 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 7 hours).

Fermentation experiments

PROTOCOL#1

• 8 ul of long term storage glycerol stock were inoculated in 8 ml of LB + suitable antibiotic in a 15 ml falcon tube and incubated at 37°C, 220 rpm in anaerobic conditions for about 16 hours. This step has been called FIRST INOCULUM.
• The culture was diluted 1:100 into 17 ml of LB + suitable antibiotic and incubated at 37°C, 220 rpm for about 6 hours (anaerobic). This step has been called SECOND INOCULUM.
• After 6 hours, 17 ml of a filter-sterilized LB + suitable antibiotic + 20% glucose was added to the culture (yielding a final 34 ml culture + 10% glucose). It was also induced in this step when required. It was incubated under the same conditions as before for 48 hours.
• After 48 hours, the pH of the pH of the culture was measured through Litmus test and the culture was centrifuged at 4°C, 9000 rpm for 15 minutes.
• Finally, the supernatant was analyzed through gas chromatography or potassium dichromate kit.

PROTOCOL#2

• 8 ul of long term storage glycerol stock were inoculated in 8 ml of LB + suitable antibiotic + 2% of glucose in a 15 ml falcon tube and incubated at 37°C in unshaked anaerobic conditions for about 24 hours. This step has been called FIRST INOCULUM.
• The pH of the culture was measured through Litmus test and then it was centrifuged at 25°C, 2500 rpm for 10 minutes.
• Supernatant was discarded and the resuspended pellet was inoculated into 30 ml of LB + suitable antibiotic + 10% of glucose in a 50 ml falcon tube and this new culture was induced (when required) and incubated at 37°C, 220 rpm for 48 hours (anaerobic). This step has been called SECOND INOCULUM.
• After 48 hours, the pH of the culture was measured again through Litmus test and the culture was centrifuged at 4°C, 9000 rpm for 15 minutes.
• Finally, the supernatant was analyzed through gas chromatography or potassium dichromate kit.

PROTOCOL#3

• 8 ul of long term storage glycerol stock were inoculated in 8 ml of LB + suitable antibiotic + 2% of glucose in a 15 ml falcon tube and incubated at 37°C, 220 rpm anaerobic conditions for about 24 hours. This step has been called FIRST INOCULUM.
• After 24 hours, 300 ul of the grown culture were inoculated into 30 ml of LB + suitable antibiotic + 10% glucose and the pH of the remaining amount of grown culture was measured through Litmus test. The new 30 ml culture was induced when required and it was incubated under the same conditions as before for 24 or 48 hours (anaerobic). This step has been called SECOND INOCULUM.
• After 24 or 48 hours, the pH of the culture was measured again through Litmus test and the culture was centrifuged at 4°C, 9000 rpm for 15 minutes.
• Finally, the supernatant was analyzed through gas chromatography or potassium dichromate kit.

Data analysis

Growth curves

The presented growth curves have all been processed as OD600_culture-OD600_broth for each time sample. OD600_broth is the medium in the same conditions as in the culture (e.g. induced with the same inducer concentration as in the culture).

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 and then this procedure was performed.

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 promoter of interest and J23101 is the reference standard promoter (taken from Anderson Promoter Collection);
• F is the blanked fluorescence of the culture, computed as F_culture-F_negative_control for each time sample, where the negative control is a non-fluorescent strain (in our experiment it is usually used or );
• ABS is the blanked absorbance (OD600) of the culture, computed as described in "Growth curves" section.

RPU measurement has the following advantages:

• 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 to satisfy are the following:

• the reporter protein must have a half life higher than the experiment duration (we use GFPmut3, , 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 validated, so the considered RPU values are in exponential growth phase, when dF/dt/ABS (proportional to the GFP synthesis rate per cell) does not vary.

Materials

• Long term glycerol stocks were stored at -80°C with a final glycerol concentration of 20%
• Antibiotics were: Ampicillin (Amp) 100 ug/ml, Kanamycin (Kan) 50 ug/ml and Chloramphenicol (Cm) 12.5 ug/ml. All of them were stored at -20°C in 1000x stocks. Amp and Kan were dissolved in water, while Cm was dissolved in ethanol 100%.
• LB medium was prepare with: 1% NaCl, 1% bactotryptone, 0.5% yeast extract. The medium was not buffered with NaOH.
• M9 supplemented medium was prepared according to: [http://openwetware.org/wiki/Knight:M9_supplemented_media Openwetware protocol].
• 3OC6-HSL (Sigma) was dissolved in water and stored at -20°C in a 2mM stock.
• aTc (Clontech) was dissolved in ethanol 50% and stored at -20°C in a 100 ug/ml stock. All the following dilutions were performed in water.
• Ready made IPTG (Sigma) was stored at -20°C in a 200mM stock.