IMPROVED PARTS

Improved Parts: list

• BBa_K300002 - Phasin (PhaP) - head domain - improvement of BBa_K208001
• BBa_K300003 - Phasin (PhaP) - internal domain - improvement of BBa_K208001

Phasins

Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by several bacteria and that accumulate as intracellular granules. Phasins are proteins that can bind these granules. The following parts are derived from the phaP gene of Ralstonia eutropha, which encodes for a phasin, engineered without the stop codon in order to support protein fusions as a head/internal domain.

Phasins can be used as affinity tags for a target protein, which can bind PHA granules allowing this way protein purification.

In literature [Banki MR et al., 2005] it has been shown that affinity tags composed by phasins assembled in tandem can increase the affinity with PHA.

The aim of our improvement of phasin already present in the Registry (<partinfo>BBa_K208001</partinfo>) was to provide a head and an internal protein domain useful to build fusion proteins.

<partinfo>BBa_K300002</partinfo> - Phasin (PhaP) - head domain

This part can be used as a N-terminal affinity tag for a target protein that has to be fused downstream. Together with <partinfo>BBa_K300003</partinfo> enables the construction of composite tags.

Because this part is a head domain, the Prefix is compatible with RFC10 and the Suffix is compatible with RFC23.

Construction

It is identical to <partinfo>BBa_K208001</partinfo>, but it lacks the stop codon in order to support protein fusions.

It has been designed as a head domain:

The Prefix sequence is 5'-GAATTCGCGGCCGCTTCTAG-3' (RFC10 Prefix)

The Suffix sequence is 5'-ACTAGTAGCGGCCGCTGCAG-3' (RFC23 Suffix)

For these reasons, a tail domain or an internal domain (compatible with RFC23) can be easily assembled downstream to create protein fusions.

To obtain this part, BioBrick <partinfo>BBa_K208001</partinfo> (provided by iGEM HQ in pSB3K3 in RFC23 standard) was PCR-amplified/mutagenized with primers:

phaP10F 5'-GCTTCTAGATGATCCTCACCCCGGAACA-3'

phaPSR: 5'-GCTACTAGTGGCAGCCGTCGTCTTCTTTG-3'

in order to delete the stop codon.

The PCR product was ran on a 1% agarose gel, gel-extracted, digested with XbaI-SpeI and ligated with <partinfo>pSB1AK3</partinfo> (previously cut with XbaI-SpeI and dephosphorylated). Positive clones were selected through digestion screening/sequencing.

This part was used to construct the following synthetic composite affinity tags:

• <partinfo>BBa_K300093</partinfo>
• <partinfo>BBa_K300094</partinfo>
• <partinfo>BBa_K300095</partinfo>
• <partinfo>BBa_K300084</partinfo>
• <partinfo>BBa_K300097</partinfo>

or alone as a head domain; in this case it has been tested through <partinfo>BBa_K300086</partinfo> measurement system.

Methods

Inoculum (into 5 ml LB+Amp) from glycerol stock of:

• <partinfo>BBa_K300086</partinfo>,
• <partinfo>BBa_K173000</partinfo> (positive control),
• <partinfo>BBa_B0031</partinfo> (negative control).

ON cultures' growth at 37°C, 220 rpm.

The following day, cultures were diluted 1:100 and let grown again for about five hours at 37°C, 220 rpm.

The optical density (O.D.) of each cell culture was than measured with TECAN Infinte F200. Samples were diluted in order to obtain the same O.D. equal to 0,02.

Then we performed a 21-hours' experiment with measurements of absorbance and green fluorescence every five minutes using TECAN Infinite F200; cultures were shaken for 15 seconds every five minutes. Each value shown below is the mean of three measurements, from GFP data that of a non-fluorescent culture (negative control) was subtracted.

Results

Raw growth curve

Raw GFP curve

Mean (dGFP/dt)/O.D. over the exponential phase (under the hypothesis that GFP half-life in fusion contructs is similar to the original one - <partinfo>BBa_E0040</partinfo>)

Discussion

All cell cultures showed a similar growth curve; doubling time was computed as described here in order to have informations about the burden due to the synthesis of such fusion proteins. It's possible to see that all doubling time are very similar; it's possible to assert that the expression of these BioBrick parts doesn't cause abnormal stress to cells.

From GFP curve it's possible to appreciate that in <partinfo>BBa_K300086</partinfo> GFP accumulation it's significantly different from that of negative control <partinfo>BBa_B0031</partinfo>. This result shows that the green fluorescent protein assembled downstream of the genetic circuit is correctly folded.

The mean protein synthesis rate was also computed over the growth exponential phase, showing an appreciable GFP production rate that is about a half of the positive control.

<partinfo>BBa_K300003</partinfo> - Phasin (PhaP) - internal domain

This part without stop codon and with Prefix and Suffix compatible with RFC23 (Silver Standard) was built in order to fully support protein fusions as an internal domain. Together with <partinfo>BBa_K300002</partinfo>, enables the construction of synthetic affinity tags containing phasins in tandem, possibly spaced by peptide linkers, as described in [Banki MR et al., 2005].

Construction

It is identical to <partinfo>BBa_K208001</partinfo>, but it lacks the stop codon in order to support protein fusions.

It has been designed as an internal domain:

The Prefix sequence is 5'-GAATTCGCGGCCGCTTCTAG-3' (RFC23 Prefix)

The Suffix sequence is 5'-ACTAGTAGCGGCCGCTGCAG-3' (RFC23 Suffix)

For these reasons, a tail domain or an internal domain (compatible with RFC23) can be easily assembled downstream this BiobBrick to create protein fusions.

To obtain this part, BioBrick <partinfo>BBa_K208001</partinfo> (provided by iGEM HQ in pSB3K3 in RFC23 standard) was PCR-amplified/mutagenized with primers:

phaPSF: 5'-GCTTCTAGAATGATCCTCACCCCGGAACA-3'

phaPSR: 5'-GCTACTAGTGGCAGCCGTCGTCTTCTTTG-3'

in order to delete the stop codon.

The PCR product was ran on a 1% agarose gel, gel-extracted, digested with XbaI-SpeI and ligated with <partinfo>pSB1A3</partinfo> (previously cut with XbaI-SpeI and dephosphorylated). Positive clones were selected through digestion screening/sequencing.

This part was used as an internal domain to construct the following synthetic composite affinity tags:

• <partinfo>BBa_K300093</partinfo>
• <partinfo>BBa_K300094</partinfo>
• <partinfo>BBa_K300084</partinfo>
• <partinfo>BBa_K300097</partinfo>