Team:TU Delft/Parts/characterization

From 2010.igem.org

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==Characterization==
==Characterization==
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Our favorite parts were also the ones best characterized. This page is a quick overview of the results.
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Our favorite parts were also the ones best characterized. This page is just a quick overview of the results. You can find a [[Team:TU_Delft/Project|complete overview of the characterization]] on the project pages.
===Favorite Parts===
===Favorite Parts===
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====BBa_K398030 and BBa_K398029====
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====[http://partsregistry.org/Part:BBa_K398029 BBa_K398029] - ALDH====
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Both Biobricks are the protein generators of Bt-ALDH. We compared the activities of bot parts, parental strain with an empty plasmid (J13002) and a natural oil-degrader (Pseudomonas putida).
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 +
[[Image:TUDelftALDH_final.jpg|600px]]
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View [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398029 BBa_K398029 in the '''parts registry''']
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Our results suggest that the recombinant strain ''E. coli'' 029A functionally express our biobrick [http://partsregistry.org/Part:BBa_K398029 BBa_K398029]. From the statistical analysis that we performed we concluded that the expression of the biobrick [http://partsregistry.org/Part:BBa_K398006 BBa_K398006] under the promoter-rbs combination [http://partsregistry.org/Part:BBa_J13002 BBa_J23100]-[http://partsregistry.org/Part:BBa_J13002 BBa_J61117] increases the dodecanal dehydrogenase activity in ''E. coli'' cell extracts 2-fold. Moreover, the enzymatic activities measured for the construct [http://partsregistry.org/Part:BBa_K398029 BBa_K398029] were equivalent to 33.98% of the ''Pseudomonas putida'' aldehyde dehydrogenase activity. 
 +
Read more about it in [https://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results our results page]
 +
 
 +
View [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398331 BBa_K398029 in the '''parts registry''']
'''Specified Components'''
'''Specified Components'''
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<partinfo>K398029 SpecifiedComponents</partinfo>
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<partinfo>BBa_K398029 SpecifiedComponents</partinfo>
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'''Designer:''' <partinfo>K398029 Designer</partinfo>
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'''Designer:''' <partinfo>BBa_K398029 Designer</partinfo>
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'''Status:''' <partinfo>K398029 Status</partinfo>
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'''Status:''' <partinfo>BBa_K398029 Status</partinfo>
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<html><span style="clear:both;display:block;width:100%;"></span></html>
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View [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398030 BBa_K398030 in the '''parts registry''']
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====[http://partsregistry.org/Part:BBa_K398331 BBa_K398331] - pCaif====
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'''Specified Components'''
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According to our results, the smallest part that we designed it's working!!!
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<partinfo>K398206 SpecifiedComponents</partinfo>
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'''Designer:''' <partinfo>K398030 Designer</partinfo>
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pCaiF a natural promoter in E. coli that is active at high cAMP levels was characterized attaching E00240 in front of our part. Different GFP production rates were found at the stationary phase showing that GFP production depends on the amount of glucose in the medium. Moreover, growth on a secondary carbon source decreases the GFP production rate.
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'''Status:''' <partinfo>K398030 Status</partinfo>
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[[Image:TU_Delft_pCaiF_final.jpg]]
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<html><span style="clear:both;display:block;width:100%;"></span></html>
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====BBa_K398206====
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Read more about our fascinating results on our [https://2010.igem.org/Team:TU_Delft#page=Project/sensing/results sensing project page]
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[[Image:TU_Delft_Emuls_assay_exp1.jpg|thumb|400px|right|Emulsification of Sudan II by the isolated proteins from the control (BBa_J13002) and AlnA (BBa_K398206) culture. Read more about the [[Team:TU_Delft/Project/solubility/results|BBa_K398206 characteristics]]]]This part contains an IPTG inducible protomor with a protein coding sequence for the production of the emulsifier AlnA. It was the main subject of our [[Team:TU_Delft/Project/solubility|solubility]] subproject.
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View [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398206 BBa_K398206 in the '''parts registry''']
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View [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398331 BBa_K398331 in the '''parts registry''']
'''Specified Components'''
'''Specified Components'''
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<partinfo>K398206 SpecifiedComponents</partinfo>
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<partinfo>K398331 SpecifiedComponents</partinfo>
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'''Designer:''' <partinfo>K398206 Designer</partinfo>
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'''Designer:''' <partinfo>K398331 Designer</partinfo>
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'''Status:''' <partinfo>K398206 Status</partinfo>
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'''Status:''' <partinfo>K398331 Status</partinfo>
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<html><span style="clear:both;display:block;width:100%;"></span></html>
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 +
====[http://partsregistry.org/wiki/index.php?title=Part:BBa_K398406 BBa_K398406] - Solvent Tolerance====
 +
The solvent tolerance cluster (BBa_K398406) was expressed on in ''E. coli'' K12. The growth of the cells was tested under different amounts of n-hexane. The results show that this part gives to the cells an growth advantage at higher n-hexane concentrations. The parental strain E. coli K12 grows really slow at 10% (v/v) of n-hexane/M9 mixture.
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====BBa_K398326====
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[[Image:TU_Delft_Solvent_tolerance.jpg|400px]]
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pCaiF is one of our Biobricks of the sensing project. It's function is to express proteins under glucose limitation conditions. In order to measure its strength we attached the GFP generator in front of it.
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Different media, and carbon source concentrations were tested in order to obtain an insight about the protein production during starvation.
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[[Image:TU_Delft_pCaiF_comp.jpg]]
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Also, it is well known that this promotor it's active during diauxic shifts, that's why we measured the GFP production using glucose as a first carbon source (concentration of 1g/L) and Potassium Laurate (5mM) as a secondary carbon source. The results that we got suggest that the part works like a charm! During glucose starvation high levels of cAMP can be found in cells, which enhances the expression of the proteins downstream of this promoter. On the other hand when the secondary carbon source is being catabolized by the cells, cAMP levels drop and thus the expression of genes regulated by pCaiF.
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Read more about these results on our [https://2010.igem.org/Team:TU_Delft#page=Project/tolerance/results survival project page].
 +
View [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398406 BBa_K398406 in the '''parts registry''']
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[[Image:TU_Delft_pCaiF_diauxic.jpg]]
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'''Specified Components'''
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<partinfo>K398406 SpecifiedComponents</partinfo>
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View [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398326 BBa_K398326 in the '''parts registry''']
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'''Designer:''' <partinfo>K398406 Designer</partinfo>
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'''Specified Components'''
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'''Status:''' <partinfo>K398406 Status</partinfo>
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<partinfo>K398326 SpecifiedComponents</partinfo>
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'''Designer:''' <partinfo>K398326 Designer</partinfo>
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====[http://partsregistry.org/wiki/index.php?title=Part:BBa_K398108 BBa_K398108] - Salt Tolerance====
 +
The growth of the bbc1 construct (BBa_K398108) was tested as stated in on [[Team:TU_Delft/Project/tolerance/characterization|the salt tolerance characterization page]].
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'''Status:''' <partinfo>K398326 Status</partinfo>
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[[Image:TU_Delft_Salt_tolerance.jpg|500px]]
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<html><span style="clear:both;display:block;width:100%;"></span></html>
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View [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398331 BBa_K398331 in the '''parts registry''']
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Read more about these results on our [https://2010.igem.org/Team:TU_Delft#page=Project/tolerance/results survival project page].
 +
 
 +
View [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398108 BBa_K398108 in the '''parts registry''']
'''Specified Components'''
'''Specified Components'''
-
<partinfo>K398331 SpecifiedComponents</partinfo>
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<partinfo>K398108 SpecifiedComponents</partinfo>
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'''Designer:''' <partinfo>K398331 Designer</partinfo>
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'''Designer:''' <partinfo>K398108 Designer</partinfo>
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'''Status:''' <partinfo>K398331 Status</partinfo>
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'''Status:''' <partinfo>K398108 Status</partinfo>
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<html><span style="clear:both;display:block;width:100%;"></span></html>
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====BBa_K398406====
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====BBa_K398206 - Emulsifier====
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The solvent tolerance cluster was expressed on E. coli K12 and tested under different amounts of n-hexane. The results suggest that this part gives to cells an advantage when there's a lot of n-hexane around. The parental strain E. coli K12 seems to grow really slow at 10% (v/v) of n-hexane/M9 mixture.
+
[[Image:TU_Delft_Emuls_assay_exp1.jpg|thumb|400px|right|Emulsification of Sudan II by the isolated proteins from the control (BBa_J13002) and AlnA (BBa_K398206) culture. Read more about the [[Team:TU_Delft/Project/solubility/results|BBa_K398206 characteristics]]]]This part contains an IPTG inducible protomor with a protein coding sequence for the production of the emulsifier AlnA. It was the main subject of our [[Team:TU_Delft/Project/solubility|solubility]] subproject.
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[[Image:TU_Delft_406_K12.jpg]]
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Whereas our E. coli 406 doesn't have any problem at all.
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View [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398206 BBa_K398206 in the '''parts registry''']
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[[Image:TU_Delft_406_406.jpg]]
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'''Specified Components'''
 +
<partinfo>K398206 SpecifiedComponents</partinfo>
 +
'''Designer:''' <partinfo>K398206 Designer</partinfo>
-
View [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398406 BBa_K398406 in the '''parts registry''']
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'''Status:''' <partinfo>K398206 Status</partinfo>
 +
<html><span style="clear:both;display:block;width:100%;"></span></html>
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'''Specified Components'''
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====Ribosome Binding Site (RBS) sequence characterization====
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<partinfo>K398406 SpecifiedComponents</partinfo>
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Read more about the [[Team:TU_Delft/Project/rbs-characterization|RBS characterization]]. We used the following 5 constructs to perform the characterization.
 +
<partinfo>BBa_K398500 SpecifiedComponents</partinfo>
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<partinfo>BBa_K398501 SpecifiedComponents</partinfo>
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<partinfo>BBa_K398502 SpecifiedComponents</partinfo>
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<partinfo>BBa_K398503 SpecifiedComponents</partinfo>
 +
<partinfo>BBa_K398504 SpecifiedComponents</partinfo>
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'''Designer:''' <partinfo>K398406 Designer</partinfo>
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[[Image:TUDelft_2010_RBS_strength_graph.PNG|thumb|400px|right|Characterization of 5 RBS sequences from the Anderson series]]
 +
Resulting RBS strenghts:
 +
{|width="300"
 +
|<b>Ribosome binding site</b>
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|<b>Mean relative strength</b>
 +
|<b>Standard deviation</b>
 +
|-
 +
|[http://partsregistry.org/Part:BBa_J61100 J61100]
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|0.020 (2.0%)
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|0.00512
 +
|-
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|[http://partsregistry.org/Part:BBa_J61101 J61101]
 +
|0.119 (11.9%)
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|0.02140
 +
|-
 +
|[http://partsregistry.org/Part:BBa_J61107 J61107]
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|0.077 (7.7%)
 +
|0.01480
 +
|-
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|[http://partsregistry.org/Part:BBa_J61117 J61117]
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|0.013 (1.3%)
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|0.00448
 +
|-
 +
|[http://partsregistry.org/Part:BBa_J61127 J61127]
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|0.065 (6.5%)
 +
|0.00660
 +
|-
 +
|[http://partsregistry.org/Part:BBa_B0032 B0032]
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|0.300 (Reference, 30%)
 +
|0.02690
 +
|}
 +
<html><br style:"clear: both;"/></html>

Latest revision as of 13:41, 27 October 2010

Characterization

Our favorite parts were also the ones best characterized. This page is just a quick overview of the results. You can find a complete overview of the characterization on the project pages.

Favorite Parts

[http://partsregistry.org/Part:BBa_K398029 BBa_K398029] - ALDH

TUDelftALDH final.jpg

Our results suggest that the recombinant strain E. coli 029A functionally express our biobrick [http://partsregistry.org/Part:BBa_K398029 BBa_K398029]. From the statistical analysis that we performed we concluded that the expression of the biobrick [http://partsregistry.org/Part:BBa_K398006 BBa_K398006] under the promoter-rbs combination [http://partsregistry.org/Part:BBa_J13002 BBa_J23100]-[http://partsregistry.org/Part:BBa_J13002 BBa_J61117] increases the dodecanal dehydrogenase activity in E. coli cell extracts 2-fold. Moreover, the enzymatic activities measured for the construct [http://partsregistry.org/Part:BBa_K398029 BBa_K398029] were equivalent to 33.98% of the Pseudomonas putida aldehyde dehydrogenase activity. Read more about it in our results page

View [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398331 BBa_K398029 in the parts registry]

Specified Components <partinfo>BBa_K398029 SpecifiedComponents</partinfo>

Designer: <partinfo>BBa_K398029 Designer</partinfo>

Status: <partinfo>BBa_K398029 Status</partinfo>

[http://partsregistry.org/Part:BBa_K398331 BBa_K398331] - pCaif

According to our results, the smallest part that we designed it's working!!!

pCaiF a natural promoter in E. coli that is active at high cAMP levels was characterized attaching E00240 in front of our part. Different GFP production rates were found at the stationary phase showing that GFP production depends on the amount of glucose in the medium. Moreover, growth on a secondary carbon source decreases the GFP production rate.

TU Delft pCaiF final.jpg

Read more about our fascinating results on our sensing project page

View [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398331 BBa_K398331 in the parts registry]

Specified Components <partinfo>K398331 SpecifiedComponents</partinfo>

Designer: <partinfo>K398331 Designer</partinfo>

Status: <partinfo>K398331 Status</partinfo>

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K398406 BBa_K398406] - Solvent Tolerance

The solvent tolerance cluster (BBa_K398406) was expressed on in E. coli K12. The growth of the cells was tested under different amounts of n-hexane. The results show that this part gives to the cells an growth advantage at higher n-hexane concentrations. The parental strain E. coli K12 grows really slow at 10% (v/v) of n-hexane/M9 mixture.

TU Delft Solvent tolerance.jpg

Read more about these results on our survival project page.

View [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398406 BBa_K398406 in the parts registry]

Specified Components <partinfo>K398406 SpecifiedComponents</partinfo>

Designer: <partinfo>K398406 Designer</partinfo>

Status: <partinfo>K398406 Status</partinfo>

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K398108 BBa_K398108] - Salt Tolerance

The growth of the bbc1 construct (BBa_K398108) was tested as stated in on the salt tolerance characterization page.

TU Delft Salt tolerance.jpg

Read more about these results on our survival project page.

View [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398108 BBa_K398108 in the parts registry]

Specified Components <partinfo>K398108 SpecifiedComponents</partinfo>

Designer: <partinfo>K398108 Designer</partinfo>

Status: <partinfo>K398108 Status</partinfo>

BBa_K398206 - Emulsifier

Emulsification of Sudan II by the isolated proteins from the control (BBa_J13002) and AlnA (BBa_K398206) culture. Read more about the BBa_K398206 characteristics
This part contains an IPTG inducible protomor with a protein coding sequence for the production of the emulsifier AlnA. It was the main subject of our solubility subproject.

View [http://partsregistry.org/wiki/index.php?title=Part:BBa_K398206 BBa_K398206 in the parts registry]

Specified Components <partinfo>K398206 SpecifiedComponents</partinfo>

Designer: <partinfo>K398206 Designer</partinfo>

Status: <partinfo>K398206 Status</partinfo>

Ribosome Binding Site (RBS) sequence characterization

Read more about the RBS characterization. We used the following 5 constructs to perform the characterization. <partinfo>BBa_K398500 SpecifiedComponents</partinfo> <partinfo>BBa_K398501 SpecifiedComponents</partinfo> <partinfo>BBa_K398502 SpecifiedComponents</partinfo> <partinfo>BBa_K398503 SpecifiedComponents</partinfo> <partinfo>BBa_K398504 SpecifiedComponents</partinfo>

Characterization of 5 RBS sequences from the Anderson series

Resulting RBS strenghts:

Ribosome binding site Mean relative strength Standard deviation
[http://partsregistry.org/Part:BBa_J61100 J61100] 0.020 (2.0%) 0.00512
[http://partsregistry.org/Part:BBa_J61101 J61101] 0.119 (11.9%) 0.02140
[http://partsregistry.org/Part:BBa_J61107 J61107] 0.077 (7.7%) 0.01480
[http://partsregistry.org/Part:BBa_J61117 J61117] 0.013 (1.3%) 0.00448
[http://partsregistry.org/Part:BBa_J61127 J61127] 0.065 (6.5%) 0.00660
[http://partsregistry.org/Part:BBa_B0032 B0032] 0.300 (Reference, 30%) 0.02690


Retrieved from "http://2010.igem.org/Team:TU_Delft/Parts/characterization"