Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts
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<table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px"> | <table border="0" align="center" width="100%"><tr><td align="justify" valign="top" style="padding:20px"> | ||
<html><p align="center"><font size="4"><b>IMPROVED PARTS</b></font></p></html><hr> | <html><p align="center"><font size="4"><b>IMPROVED PARTS</b></font></p></html><hr> | ||
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<tr><td width="100%"> | <tr><td width="100%"> | ||
- | < | + | |
- | + | <table class="cont" border="2" width="100%" align="center"> | |
- | <td align="center" width="25% | + | <tr> |
- | [https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization Characterization] | + | <td align="center" width="25%"> |
+ | [https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization Return to Characterization] | ||
</td> | </td> | ||
- | < | + | <th align="center" width="25%"> |
- | [https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/NewParts New Parts] | + | [https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/NewParts New Parts] |
- | </ | + | </th> |
- | < | + | <th align="center" width="25%"> |
- | [https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts Improved Parts] | + | [https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts Improved Parts] |
- | </ | + | </th> |
- | < | + | <th align="center" width="25%"> |
- | [https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry Existing Parts from the Registry]<br> | + | [https://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization/ExistingPartsRegistry Existing Parts from the Registry] |
+ | </th> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | |||
+ | =Improved Parts: list= | ||
+ | |||
+ | * [[Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts #BBa_K300002 - Phasin (PhaP) - head domain|BBa_K300002 - Phasin (PhaP) - head domain - improvement of BBa_K208001]] | ||
+ | * [[Team:UNIPV-Pavia/Parts/Characterization/RebExistingParts #BBa_K300003 - Phasin (PhaP) - internal domain|BBa_K300003 - Phasin (PhaP) - internal domain - improvement of BBa_K208001]] | ||
+ | |||
+ | <br> | ||
+ | ---- | ||
+ | <br> | ||
+ | |||
</td></tr> | </td></tr> | ||
- | < | + | <tr><td> |
- | <tr><td>< | + | =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=== | ||
+ | <table align="center"> | ||
+ | <tr> | ||
+ | <td>[[Image:UNIPV10_pTET_Phas_ASB.png|thumb|300px|Raw growth curve]]</td> | ||
+ | <td>[[Image:UNIPV10_pTET_Phas_GFP.png|thumb|300px|Raw GFP curve]]</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <table align="center"> | ||
+ | <tr> | ||
+ | <td>[[Image:UNIPV10_pTET_Phas_BAR.png|thumb|300px|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>)]]</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <table border="1" align="center"> | ||
+ | <tr align="center"> | ||
+ | <th>Culture</th><th>Doubling time [min.] ± std error</th> | ||
+ | </tr> | ||
+ | <tr align="center"> | ||
+ | <td><partinfo>BBa_K173000</partinfo></td><td>76.3336 ± 1.4362</td> | ||
+ | </tr> | ||
+ | <tr align="center"> | ||
+ | <td><partinfo>BBa_K300086</partinfo></td><td>73.6685 ± 1.6245</td> | ||
+ | </tr> | ||
+ | <tr align="center"> | ||
+ | <td><partinfo>BBa_B0031</partinfo></td><td>70.8421 ± 2.2181</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | ===Discussion=== | ||
+ | All cell cultures showed a similar growth curve; 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 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> | ||
+ | |||
+ | </td> | ||
+ | </tr> | ||
+ | </table> |
Latest revision as of 03:25, 28 October 2010
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