Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts

From 2010.igem.org

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m (BBa_K300003 - Phasin (PhaP) - internal domain)
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=Pasins=
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=<partinfo>BBa_K300002</partinfo> - Phasin (PhaP) - head domain=
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Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by diverse bacteria and that accumulate as intracellular granules. Phasins are proteins that can bind these granules.
Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by diverse bacteria and that accumulate as intracellular granules. Phasins are proteins that can bind these granules.
This part is the phaP gene from ''Ralstonia eutropha'', which encodes for a phasin, without stop codon in order to support protein fusions as a head domain.
This part is the phaP gene from ''Ralstonia eutropha'', which encodes for a phasin, without stop codon in order to support protein fusions as a head domain.
It can be used as a N-terminal affinity tag for a target protein that has to be fused downstream of the phasin. The tagged protein adhere to the surface of the PHA granules via the phasin tag, which enables the target protein purification.
It can be used as a N-terminal affinity tag for a target protein that has to be fused downstream of the phasin. The tagged protein adhere to the surface of the PHA granules via the phasin tag, which enables the target protein purification.
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In literature [Banki MR et al.] it has been shown that affinity tags composed by phasins assembled in tandem can increase the affinity with PHA. This part, together with BBa_K300003 enables the construction of such composite tags.
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In literature [Banki MR et al.] it has been shown that affinity tags composed by phasins assembled in tandem can increase the affinity with PHA.
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==<partinfo>BBa_K300002</partinfo> - Phasin (PhaP) - head domain==
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This part, together with BBa_K300003 enables the construction of such composite tags.
Because this part is a head domain, the Prefix is compatible with RFC10 and the Suffix is compatible with RFC23.
Because this part is a head domain, the Prefix is compatible with RFC10 and the Suffix is compatible with RFC23.
This part has been tested through <partinfo>BBa_K300086</partinfo> measurement system.
This part has been tested through <partinfo>BBa_K300086</partinfo> measurement system.
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==Methods==
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===Methods===
Inoculum (into 5 ml LB+Amp) from glycerol stock of:
Inoculum (into 5 ml LB+Amp) from glycerol stock of:
*<partinfo>BBa_K300086</partinfo>
*<partinfo>BBa_K300086</partinfo>
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Than we performed a 21 hours' experiment with measurements of absorbance and green fluorescence every five minutes with TECAN Infinite F200. Each value shown is the mean of three measurements, from GFP data that of a non-fluorescent culture (negative control) was subtracted; cultures were shaken for 15 seconds every five minutes.
Than we performed a 21 hours' experiment with measurements of absorbance and green fluorescence every five minutes with TECAN Infinite F200. Each value shown is the mean of three measurements, from GFP data that of a non-fluorescent culture (negative control) was subtracted; cultures were shaken for 15 seconds every five minutes.
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==Results==
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===Results===
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====Discussion====
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===Discussion===
All cell cultures showed a similar growth curve and doubling time was computed as described [[Team:UNIPV-Pavia/Parts/Characterization#Doubling_time_evaluation|here]] in order to have informations about the burden due to 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 the cells.
All cell cultures showed a similar growth curve and doubling time was computed as described [[Team:UNIPV-Pavia/Parts/Characterization#Doubling_time_evaluation|here]] in order to have informations about the burden due to 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 the cells.
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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.
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.
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=<partinfo>BBa_K300003</partinfo> - Phasin (PhaP) - internal domain=
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==<partinfo>BBa_K300003</partinfo> - Phasin (PhaP) - internal domain==
Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by diverse bacteria and that accumulate
Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by diverse bacteria and that accumulate
as intracellular granules. Phasins are proteins that can bind these granules.
as intracellular granules. Phasins are proteins that can bind these granules.

Revision as of 22:45, 26 October 2010

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Contents

Pasins

Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by diverse bacteria and that accumulate as intracellular granules. Phasins are proteins that can bind these granules. This part is the phaP gene from Ralstonia eutropha, which encodes for a phasin, without stop codon in order to support protein fusions as a head domain. It can be used as a N-terminal affinity tag for a target protein that has to be fused downstream of the phasin. The tagged protein adhere to the surface of the PHA granules via the phasin tag, which enables the target protein purification. In literature [Banki MR et al.] it has been shown that affinity tags composed by phasins assembled in tandem can increase the affinity with PHA.

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

This part, together with BBa_K300003 enables the construction of such composite tags.

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

This part 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)

They were let grow ON at +37°C, 220 rpm.

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

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.

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

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>)
CultureDoubling time [min.]
<partinfo>BBa_K173000</partinfo>77
<partinfo>BBa_K300086</partinfo>74
<partinfo>BBa_B0031</partinfo>69

Discussion

All cell cultures showed a similar growth curve and doubling time was computed as described here in order to have informations about the burden due to 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 the cells.

In 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 the right folding of the green fluorescent protein assembled downstream of the genetic circuit.

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

Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by diverse bacteria and that accumulate as intracellular granules. Phasins are proteins that can bind these granules.

This part is the phaP gene from Ralstonia eutropha, which encodes for a phasin, without stop codon and with Prefix and Suffix compatible with RFC23 (Silver Standard) in order to fully support protein fusions as an internal domain.

Phasins can be used as affinity tags for a target protein, which can bind PHA granules via the phasin tag. It has been shown that mutiple phasins in tandem can increase the affinity with PHA. This part, together with <partinfo>BBa_K300002</partinfo>, enables the construction of synthetic affinity tags based on phasins in tandem, possibly spaced by peptide linkers, as described in [Banki MR et al.].


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