Difference between revisions of "Part:BBa E1010:Experience"

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<partinfo>BBa_E1010 3</partinfo>
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<partinfo>BBa_E1010 AddReview 3 </partinfo>
 
<I>DTU_igem_2010</I>
 
<I>DTU_igem_2010</I>
 
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'''Characterization of RFP BBa_E1010'''
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'''Characterization of RFP BBa_E1010'''<br>
 
We have characterized RFP BBa_E1010 in two different chassis to test the compatibility and the possible range of expressions before limitations in the cell metabolism.
 
We have characterized RFP BBa_E1010 in two different chassis to test the compatibility and the possible range of expressions before limitations in the cell metabolism.
  
−
'''Method'''
+
'''Method'''<br>
 
We have made constructs with a synthetic promoter library (SPL) in front of the E1010, by using BBa_I03507 and the psB3T5.  For information on design of an SPL compatible with the BB standard see [http://bbf.openwetware.org/RFC.html#BBF_RFC_63:_DTU_Synthetic_Promoter_Library_Standard BBF RFC63].
 
We have made constructs with a synthetic promoter library (SPL) in front of the E1010, by using BBa_I03507 and the psB3T5.  For information on design of an SPL compatible with the BB standard see [http://bbf.openwetware.org/RFC.html#BBF_RFC_63:_DTU_Synthetic_Promoter_Library_Standard BBF RFC63].
 
We have benchmarked the relative promoter strength range achieved from the SPL to the standard promoter BBa_J23101, by calculating the relative promoter strength in vivo as suggested in [http://bbf.openwetware.org/RFC.html#BBF_RFC_19:_Measuring_the_Activity_of_BioBrick.E2.84.A2_Promoters_Using_an_In_Vivo_Reference_Standard BBF RFC 19]. For further explanation on methods see our [http://2010.igem.org/Team:DTU-Denmark iGEM_DTU_2010 wiki].
 
We have benchmarked the relative promoter strength range achieved from the SPL to the standard promoter BBa_J23101, by calculating the relative promoter strength in vivo as suggested in [http://bbf.openwetware.org/RFC.html#BBF_RFC_19:_Measuring_the_Activity_of_BioBrick.E2.84.A2_Promoters_Using_an_In_Vivo_Reference_Standard BBF RFC 19]. For further explanation on methods see our [http://2010.igem.org/Team:DTU-Denmark iGEM_DTU_2010 wiki].
  
−
'''Results'''
+
'''Results'''<br>
 
We show that the RFP E1010 can be expressed with the following results:
 
We show that the RFP E1010 can be expressed with the following results:
 
  * In XL1blue with an RPU range form 0 to at least 1,13 RPU.
 
  * In XL1blue with an RPU range form 0 to at least 1,13 RPU.
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[[Image:Graph4 DH5a.png|center|thumb|400px|]]
 
[[Image:Graph4 DH5a.png|center|thumb|400px|]]
 
[[Image:Graph5 DH5a.png|center|thumb|400px|]]
 
[[Image:Graph5 DH5a.png|center|thumb|400px|]]
 +
 +
<table cellpadding="2" border="1px" cellspacing="0" align="center" width="70%">
 +
<caption><p align="justify"><b>Table 1</b></p></caption>
 +
<thead>
 +
<td align="center"><b>Construct</b></td><td align="center"><b>Specific Activity</b></td><td align="center"><b>RPU</b></td>
 +
</thead></tr>
 +
<tr>
 +
<td align="left">BBa_J23101</td><td align="right">0.0795</td><td align="right">1.00</td>
 +
</tr>
 +
<tr>
 +
<td align="left">SPL_RFP01</td><td align="right">0.00578</td><td align="right">0.0727</td>
 +
</tr>
 +
<tr>
 +
<td align="left">SPL_RFP02</td><td align="right">0.0418</td><td align="right">0.526</td>
 +
</tr>
 +
<tr>
 +
<td align="left">SPL_RFP03</td><td align="right">0.0612</td><td align="right">0.770</td>
 +
</tr>
 +
<tr>
 +
<td align="left">SPL_RFP04</td><td align="right">-0.00027</td><td align="right">-0.00340</td>
 +
</tr>
 +
<tr>
 +
<td align="left">SPL_RFP05</td><td align="right">0.0418</td><td align="right">0.526</td>
 +
</tr>
 +
<tr>
 +
<td align="left">SPL_RFP06</td><td align="right">0.0856</td><td align="right">1.08</td>
 +
</tr>
 +
<tr>
 +
<td align="left">SPL_RFP07</td><td align="right">0.0134</td><td align="right">0.168</td>
 +
</tr>
 +
<tr>
 +
<td align="left">SPL_RFP08</td><td align="right">0.0534</td><td align="right">0.672</td>
 +
</tr>
 +
<tr>
 +
<td align="left">SPL_RFP09</td><td align="right">0.0638</td><td align="right">0.803</td>
 +
</tr>
 +
<tr>
 +
<td align="left">SPL_RFP10</td><td align="right">0.00260</td><td align="right">0.0327</td>
 +
</tr>
 +
<tr>
 +
<td align="left">SPL_RFP11</td><td align="right">0.0900</td><td align="right">1.13</td>
 +
</tr>
 +
<tr>
 +
<td align="left">SPL_RFP12</td><td align="right">0.0600</td><td align="right">0.755</td>
 +
</tr>
 +
<tr>
 +
<td align="left">SPL_RFP13</td><td align="right">0.0754</td><td align="right">0.949</td>
 +
</tr>
 +
<tr>
 +
<td align="left">SPL_RFP14</td><td align="right">0.00795</td><td align="right">0.100</td>
 +
</tr>
 +
<tr>
 +
<td align="left">SPL_RFP16</td><td align="right">0.00959</td><td align="right">0.121</td>
 +
</tr>
 +
</table>
 +
 +
 +
<table cellpadding="2" border="1px" cellspacing="0" align="center" width="70%">
 +
<caption><p align="justify"><b>Table 2 shows the specific activities and RPUs calculated for all the SPL constructs run in the second set of measurements in BioLector</b></p></caption>
 +
<thead>
 +
<td align="center"><b>Construct</b></td><td align="center"><b>Specific Activity</b></td><td align="center"><b>RPU</b></td>
 +
</thead></tr>
 +
<tr>
 +
<td align="left">BBa_J23101</td><td align="right">0.0712</td><td align="right">1.00</td>
 +
</tr>
 +
<tr>
 +
<td align="left">SPL_RFP-D01</td><td align="right">0.0715</td><td align="right">1.00</td>
 +
</tr>
 +
<tr>
 +
<td align="left">SPL_RFP-D02</td><td align="right">0.0959</td><td align="right">1.35</td>
 +
</tr>
 +
<tr>
 +
<td align="left">SPL_RFPD-03</td><td align="right">0.0781</td><td align="right">1.10</td>
 +
</tr>
 +
<tr>
 +
<td align="left">SPL_RFPD-04</td><td align="right">0.0531</td><td align="right">0.746</td>
 +
</tr>
 +
<tr>
 +
<td align="left">SPL_RFPD-05</td><td align="right">0.0732</td><td align="right">1.03</td>
 +
</tr>
 +
<tr>
 +
<td align="left">SPL_RFPD-06</td><td align="right">0.0552</td><td align="right">0.775</td>
 +
</tr>
 +
<tr>
 +
<td align="left">SPL_RFPD-07</td><td align="right">0.0358</td><td align="right">0.503</td>
 +
</tr>
 +
<tr>
 +
<td align="left">SPL_RFPD-08</td><td align="right">0.0668</td><td align="right">0.938</td>
 +
</tr>
 +
<tr>
 +
<td align="left">SPL_RFPD-09</td><td align="right">0.0254</td><td align="right">0.357</td>
 +
</tr>
 +
<tr>
 +
<td align="left">SPL_RFPD-10</td><td align="right">0.0165</td><td align="right">0.231</td>
 +
</tr>
 +
<tr>
 +
<td align="left">SPL_RFPD-11</td><td align="right">-0.00284</td><td align="right">-0.399</td>
 +
</tr>
 +
<tr>
 +
<td align="left">SPL_RFPD-12</td><td align="right">0.00276</td><td align="right">0.0387</td>
 +
</tr>
 +
<tr>
 +
<td align="left">SPL_RFPD-13</td><td align="right">0.0776</td><td align="right">1.09</td>
 +
</tr>
 +
<tr>
 +
<td align="left">SPL_RFPD-14</td><td align="right">0.0018</td><td align="right">0.0253</td>
 +
</tr>
 +
<tr>
 +
<td align="left">SPL_RFPD-15</td><td align="right">0.00302</td><td align="right">0.0424</td>
 +
</tr>
 +
</table>
 +
  
 
'''Conclusions and Discussion'''
 
'''Conclusions and Discussion'''

Revision as of 03:26, 28 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.

RANDOM SEQUENCE FOUND WITHIN PART

CGCTGATAGTGCTAGTGTAGATCGC is found after the RFP stop codon and before the BioBricks suffix. Should not affect transcription or translation of RFP, but good to keep note of it especially in analyzing sequencing results. (KP of siGEM)

Applications of BBa_E1010

User Reviews

UNIQe287e6bf3dadc0e2-partinfo-00000000-QINU

•••

DTU_igem_2010

Characterization of RFP BBa_E1010
We have characterized RFP BBa_E1010 in two different chassis to test the compatibility and the possible range of expressions before limitations in the cell metabolism.

Method
We have made constructs with a synthetic promoter library (SPL) in front of the E1010, by using BBa_I03507 and the psB3T5. For information on design of an SPL compatible with the BB standard see [http://bbf.openwetware.org/RFC.html#BBF_RFC_63:_DTU_Synthetic_Promoter_Library_Standard BBF RFC63]. We have benchmarked the relative promoter strength range achieved from the SPL to the standard promoter BBa_J23101, by calculating the relative promoter strength in vivo as suggested in [http://bbf.openwetware.org/RFC.html#BBF_RFC_19:_Measuring_the_Activity_of_BioBrick.E2.84.A2_Promoters_Using_an_In_Vivo_Reference_Standard BBF RFC 19]. For further explanation on methods see our [http://2010.igem.org/Team:DTU-Denmark iGEM_DTU_2010 wiki].

Results
We show that the RFP E1010 can be expressed with the following results: 

* In XL1blue with an RPU range form 0 to at least 1,13 RPU.
* In DHA5&alpha with an RPU range from 0 to 1,35 RPU.
Graph2 XL1BLUE.png
Graph3 XL1BLUE.png
Graph4 DH5a.png
Graph5 DH5a.png

<thead>

</thead></tr>

Table 1
ConstructSpecific ActivityRPU
BBa_J231010.07951.00
SPL_RFP010.005780.0727
SPL_RFP020.04180.526
SPL_RFP030.06120.770
SPL_RFP04-0.00027-0.00340
SPL_RFP050.04180.526
SPL_RFP060.08561.08
SPL_RFP070.01340.168
SPL_RFP080.05340.672
SPL_RFP090.06380.803
SPL_RFP100.002600.0327
SPL_RFP110.09001.13
SPL_RFP120.06000.755
SPL_RFP130.07540.949
SPL_RFP140.007950.100
SPL_RFP160.009590.121


<thead>

</thead></tr>

Table 2 shows the specific activities and RPUs calculated for all the SPL constructs run in the second set of measurements in BioLector
ConstructSpecific ActivityRPU
BBa_J231010.07121.00
SPL_RFP-D010.07151.00
SPL_RFP-D020.09591.35
SPL_RFPD-030.07811.10
SPL_RFPD-040.05310.746
SPL_RFPD-050.07321.03
SPL_RFPD-060.05520.775
SPL_RFPD-070.03580.503
SPL_RFPD-080.06680.938
SPL_RFPD-090.02540.357
SPL_RFPD-100.01650.231
SPL_RFPD-11-0.00284-0.399
SPL_RFPD-120.002760.0387
SPL_RFPD-130.07761.09
SPL_RFPD-140.00180.0253
SPL_RFPD-150.003020.0424


Conclusions and Discussion

;

Antiquity

This review comes from the old result system and indicates that this part did not work in some test.

No review score entered. Nkessler

We successfully used this part for a read out system, e.g. in BBa_K389016. Additionally we compared it with a luciferase: BBa_K389004.

UNIQe287e6bf3dadc0e2-partinfo-00000006-QINU