Difference between revisions of "Part:BBa K300002:Experience"

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(→‎Applications of BBa_K300002)
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===Applications of BBa_K300002===
 
===Applications of BBa_K300002===
 +
===Applications of BBa_K300097===
 +
====Methods====
 +
Inoculum (into 5 ml LB+Amp) from glycerol stock of:
 +
*<partinfo>BBa_K300086</partinfo>
 +
*<partinfo>BBa_K300088</partinfo>
 +
*<partinfo>BBa_K300090</partinfo>
 +
*<partinfo>BBa_K300099</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====
 +
<div align="center">
 +
<table border="0">
 +
<tr>
 +
<td>[[Image:UNIPV10_pTET_c_ASB.png|thumb|300px|Raw growth curve]]</td>
 +
<td>[[Image:UNIPV10_pTET_c_GFP.png|thumb|300px|Raw GFP curve]]</td>
 +
</tr>
 +
</table>
 +
<table>
 +
<tr>
 +
<td>[[Image:UNIPV10_pTET_c_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">
 +
<tr align="center">
 +
<th>Culture</th><th>Doubling time [min.]</th>
 +
</tr>
 +
<tr align="center">
 +
<td><partinfo>BBa_K173000</partinfo></td><td>77</td>
 +
</tr>
 +
<tr align="center">
 +
<td><partinfo>BBa_K300086</partinfo></td><td>74</td>
 +
</tr>
 +
<tr align="center">
 +
<td><partinfo>BBa_K300088</partinfo></td><td>75</td>
 +
</tr>
 +
<tr align="center">
 +
<td><partinfo>BBa_K300090</partinfo></td><td>76</td>
 +
</tr>
 +
<tr align="center">
 +
<td><partinfo>BBa_K300099</partinfo></td><td>76</td>
 +
</tr>
 +
<tr align="center">
 +
<td><partinfo>BBa_B0031</partinfo></td><td>69</td>
 +
</tr>
 +
</table>
 +
</div>
 +
====Discussion====
 +
All cell cultures showed a similar growth curve and doubling time was computed as described [http://2010.igem.org/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.
 +
 +
In GFP curve it's possible to appreciate that in <partinfo>BBa_K300086</partinfo>, <partinfo>BBa_K300088</partinfo>, <partinfo>BBa_K300090</partinfo>, <partinfo>BBa_K300099</partinfo> GFP accumulation it's very similar and it's significantly different from that of negative control <partinfo>BBa_B0031</partinfo>. These results show 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 again an appreciable GFP production rate that is about a half of the positive control.
  
 
===User Reviews===
 
===User Reviews===

Revision as of 14:23, 26 October 2010

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K300002

Applications of BBa_K300097

Methods

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

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 -BBa_E0040)
CultureDoubling time [min.]
BBa_K17300077
BBa_K30008674
BBa_K30008875
BBa_K30009076
BBa_K30009976
BBa_B003169

Discussion

All cell cultures showed a similar growth curve and doubling time was computed as described [http://2010.igem.org/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.

In GFP curve it's possible to appreciate that in BBa_K300086, BBa_K300088, BBa_K300090, BBa_K300099 GFP accumulation it's very similar and it's significantly different from that of negative control BBa_B0031. These results show 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 again an appreciable GFP production rate that is about a half of the positive control.

User Reviews

UNIQdaaff311d4d0055f-partinfo-00000012-QINU

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UNIPV-Pavia iGEM 2010

This part was used as a head domain alone and to construct the following synthetic composite affinity tags:


UNIQdaaff311d4d0055f-partinfo-00000019-QINU