Generator

Part:BBa_K173004:Experience

Designed by: Lorenzo Pasotti, Susanna Schiavi, Letizia Diamante, Paolo Magni   Group: iGEM09_UNIPV-Pavia   (2009-10-15)
Revision as of 00:03, 22 October 2009 by Susanna (Talk | contribs)

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Applications of BBa_K173004

BBa_K173004/BBa_I732019 - beta-galactosidase protein generator - UNIPV-Pavia (Test performed by S. Schiavi, L. Pasotti)

Description

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

We built up BBa_K173004. It is a twin of BBa_I732019, 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 (BBa_I732005), 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

Compatibility: E. coli TOP10 in pSB1AK3

50 ul of BBa_K173004, BBa_K173006, BBa_V1022 (positive control) and BBa_B0032 were plated on LB + Amp + X-Gal + IPTG plates (except for BBa_V1022 for which non selective LB was used). Plated bacteria were incubated at 37°C for about 16 hours and then a picture of the plates was taken.

To prepare these plates, 20 ul of X-Gal 40 mg/ml in DMF and 20 ul of ready made IPTG were diluted in 60 ul of SOC medium and spread on a LB agar plate.

BBa_K173006
BBa_B0032
BBa_V1022
BBa_K173004
Conclusions

We improved the existing BBa_I732019 part building up BBa_K173004 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 BBa_K173005 upstream, which provides a promoter strength of 1.58 [1.56 ; 1.67] RPU in LB medium. BBa_K173005 can be considered as a measurement system for BBa_K173004. It has been tested in pSB1AK3 vector.

Surprisingly, as reported in the 4th picture, in the plate with the promoterless protein generator blue colour can be seen. It may be due to i) spurious transcription of the protein generator in the high copy number plasmid pSB1AK3 or ii) to the recombination occurred between plasmidic lacZ and genomic lacZdeltaM15, in which the working lacZ was integrated in E. coli genome under the control of lac promoter. This phenomenon has still to be studied. Caltech iGEM 2008 team reported this phenomenon in a similar protein generator, in which beta-gal assay was performed.


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).


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].
  • X-Gal (Sigma) was stored at -20°C in a 40mg/ml stock
  • Ready made IPTG (Sigma) was stored at -20°C in a 200mM stock.


User Reviews

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