• Home
• The Project
  • Motivation
  • Solution
  • Results & Conclusions
  • References
• Materials & Methods
  • Measurements
  • Instruments
  • Protocols
• Software Developed
• Parts
  • Submitted
  • Characterization
• The Team
• Notebook
  • Week-book
  • Freezer Management
• Biological Safety
• Pavia
  • History
  • University
  • Gallery
• Sponsors
• Gallery
• Team Locations

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

BBa_K173007 - aTc inducible device with J23100 promoter

Description

This is an aTc sensing device.

BBa_J23100 promoter drives the constitutive production of tetR repressor (BBa_C0040), which inhibits tetR promoter (BBa_R0040) 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.

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).

BBa_K173011 - aTc inducible device with J23118 promoter

BBa_K173010 - lactose/IPTG inducible device with J23118 promoter

Re-built existing parts

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_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.

Experiment Na+ 0M

Motivation

In our opinion the working principle of the antiporter Na+/H+ channel described in [1-2-3-4] 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

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

Methods

Results

Comments

BBa_K112808 - Enterobacteria phage T4 Lysis Device

BBa_R0011 - Plac hybrid promoter

Growth conditions

For every culture we tested:

• 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 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).

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;
• 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].
• 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.