Part:BBa_I732019:Experience
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Applications of BBa_I732019
This part was inconsistent in 2008 DNA Distribution, according to iGEM QC. The UNIPV-Pavia iGEM 2009 team built up BBa_K173004 as a twin of this part and characterized it. here we reporter the characterization results.
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.
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.
Materials
- Long term glycerol stocks were stored at -80°C with a final glycerol concentration of 20%.
- LB medium was prepare with: 1% NaCl, 1% bactotryptone, 0.5% yeast extract. The medium was not buffered with NaOH.
- 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.
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