Team:UNIPV-Pavia/Existing

• 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

Our new parts

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

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

Existing parts: sequence debugging

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

Growth curves for 3OC6HSL in M9

Growth curves for 3OC6HSL in LB

(dGFP/dt)/OD in LB and M9

Charachterization of part in M9

Charachterization of part in LB

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 1nM and 10nM.

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

from iGEM 2008 NYMU-Taipei

We received the BioBrick measurement system (which has a GFP protein generator downstream) of from iGEM HQ in September (it is called ). The bacterial strain that contained the plasmid was NEB 10-beta, so we decided to 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 into 4ml of LB + Amp and incubated overnight at 37°C, 220 rpm. We did the same for TOP10 with and 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

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

Comments

As expected 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 into 4ml of LB + Amp and incubated overnight at 37°C, 220 rpm. We did the same for TOP10 with and 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

Absorbance	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

Actually a lysis device such as is not part of "Ethanol? Whey not!" project. Anyway, we found very interesting to characterize the activity of this BioBrick and we decided to do it by building up a specific measurement system: , in which we can modulate the expression of the lysis genes through inducible device. During this summer we have also characterized in standard units, so the characterization of the lysis device is device-independent.

receiver device controls the expression of holin () and lysozyme () genes from T4 bacteriophage. Holins form pores in the inner membrane of bacteria, while lysozymes degrade the peptidoglycan layer, passing through these pores and performing the lysis of the bacterial cell. This device also contains a constitutive expression of an antiholin (), which inhibits holin multimer formation.

Lysis 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 ().

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

Growth curves for BBa_K173025 in LB

(dGFP/dt)/OD in LB and LB

Induction curve of BBa_K173025 in LB

Conclusions