Team:EPF Lausanne/Project parts

From 2010.igem.org

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ork in ''E. coli'', which is very useful for our sets of experiments.
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{{EPFL_2010_2}}
{{EPFL_2010_2}}
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<html>
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<div CLASS="EPFL_content">
<div CLASS="EPFL_content">
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[[Image:Asaia Parts 2.png|center|200px|caption]]
 
=Cloning=
=Cloning=
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We performed all the cloning in ''E.coli'', and did experiments on Asaia transformation, growth and resistance to antibiotics in parallel.
+
[[Image:Eat.png|center|500px|caption]]
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As ''E. coli'' origin does not work in Asaia, we added an origin that was compatible with this bacteria (which was recovered from a GFP-plasmid transformed into the Asaia we received from Italy). On the opposite, Asaia's origin does work in ''E. coli'', which is very useful for our sets of experiments.
+
We performed all the cloning in ''E.coli'', and ran experiments on ''Asaia'' transformation, growth, and antibiotic resistance in parallel.
 +
As the ''E. coli'' origin of replication does not work in ''Asaia'', we added an origin that is compatible with ''Asaia'' (which was recovered from a GFP-plasmid transformed into the ''Asaia'' we received from Italy). Conversely, ''Asaia's'' origin of replication works in ''E. coli'', which is very useful as all cloning can be performed using standards developed for ''E. coli''.
= BioBricks =
= BioBricks =
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All our parts are in the pSB1C3 backbone vector (abbreviate C3). When you click on the part's name, you are redirected to the partsregistry corresponding page.  
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[[Image:Asaia Parts 2.png|right|200px|caption]]
 +
All our parts are in the pSB1C3 backbone vector (abbreviate C3).
First the legend:
First the legend:
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*Ori = Origin of replication for Asaia and ''E. coli''
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*Ori = Origin of replication for ''Asaia'' and ''E. coli''
*S = Strong Promoter
*S = Strong Promoter
*W = Weak Promoter
*W = Weak Promoter
Line 39: Line 42:
*Tet = Resistance to Tetracycline
*Tet = Resistance to Tetracycline
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We made basic parts to build the complexest one.
 
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[http://partsregistry.org/Part:BBa_K320000 *C3 + Ori] We made this plasmid because we needed an origin of replication that works in Asaia. After experiences we concluded that it also work with ''E. coli''.
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This are the first and basic constructs we made. Just click on the part to get more information.
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[http://partsregistry.org/Part:BBa_K320001 *C3 + Immu] The immunotoxin is the main part of the project, we obtained it by synthetization. For a better understanding, go [https://2010.igem.org/Team:EPF_Lausanne/Project_immuno there].
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<html>
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[http://partsregistry.org/Part:BBa_K320002 *C3 + S] We made a new strong promoter permitting use to test the expression of the immunotoxine. We called it strong because we used the constitutive sequence which should give the biggets level of expression.
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<img src="https://static.igem.org/mediawiki/2010/a/a5/First_three.png" usemap = #example border=0>
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<map name=example>
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<area shape=Rect Coords=27,57,150,105 Href="http://partsregistry.org/Part:BBa_K320000">
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<area shape=Rect Coords=39,162,168,222 Href="http://partsregistry.org/Part:P1002">
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<area shape=Rect Coords=36,264,162,321 Href="http://partsregistry.org/Part:P1003">
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<area shape=Rect Coords=39,372,165,462 Href="http://partsregistry.org/Part:P1005">
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<area shape=Rect Coords=492,54,765,117 Href="http://partsregistry.org/Part:BBa_K320011">
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<area shape=Rect Coords=510,189,780,255 Href="http://partsregistry.org/Part:BBa_K320004">
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<area shape=Rect Coords=492,318,762,390 Href="http://partsregistry.org/Part:BBa_K320003">
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<area shape=Rect Coords=24,651,198,771 Href="http://partsregistry.org/Part:BBa_K320002">
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<area shape=Rect Coords=24,810,189,918 Href="http://partsregistry.org/Part:BBa_K320010">
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<area shape=Rect Coords=21,969,186,1035 Href="http://partsregistry.org/Part:BBa_K320001">
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<area shape=Rect Coords=21,1092,186,1140 Href="http://partsregistry.org/Part:BBa_K320015">
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<area shape=Rect Coords=22,1176,168,1236 Href="http://partsregistry.org/Part:BBa_K320016">
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<area shape=Rect Coords=24,1260,183,1323 Href="http://partsregistry.org/Part:BBa_E0020">
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<area shape=Rect Coords=489,669,795,792 Href="http://partsregistry.org/Part:BBa_K320006">
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<area shape=Rect Coords=492,861,816,972 Href="http://partsregistry.org/Part:BBa_K320007">
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<area shape=Rect Coords=501,1173,819,1299 Href="http://partsregistry.org/Part:BBa_K320005">
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</map>
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[http://partsregistry.org/Part:BBa_K320010 *C3 + W] This promoter has been made to have a second level of expression for our proteins, which is lower than the strong one.
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</html>
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We made the following BioBricks from the aboves :
 
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[[Image:Asaia_Parts.png|300px|right|Building the biobricks...]]
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These are the BioBricks we obtained by combining the parts from above.  
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[http://partsregistry.org/Part:BBa_K320003 *C3 + Ori + Tet]
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<html>
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[http://partsregistry.org/Part:BBa_K320004 *C3 + Ori + Kan]
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<img src="https://static.igem.org/mediawiki/2010/5/5e/Second_three.png" usemap = #second_tree border=0>
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<map name=second_tree>
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<area shape=Rect Coords=26,26,174,60 Href="http://partsregistry.org/Part:BBa_K320011">
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<area shape=Rect Coords=18,100,186,138 Href="http://partsregistry.org/Part:BBa_K320004">
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<area shape=Rect Coords=22,180,182,210 Href="http://partsregistry.org/Part:BBa_K320003">
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<area shape=Rect Coords=24,376,198,446 Href="http://partsregistry.org/Part:BBa_K320006">
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<area shape=Rect Coords=22,482,192,552 Href="http://partsregistry.org/Part:BBa_K320007">
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<area shape=Rect Coords=22,662,206,730 Href="http://partsregistry.org/Part:BBa_K320005">
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<area shape=Rect Coords=350,144,686,214 Href="http://partsregistry.org/Part:BBa_K320009">
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<area shape=Rect Coords=348,262,694,332 Href="http://partsregistry.org/Part:BBa_K320008">
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<area shape=Rect Coords=346,474,864,540 Href="http://partsregistry.org/Part:BBa_K320014">
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</map>
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[http://partsregistry.org/Part:BBa_K320005 *C3 + S + CFP]
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</html>
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[http://partsregistry.org/Part:BBa_K320006 *C3 + S + Immu]
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[http://partsregistry.org/Part:BBa_K320007 *C3 + S + P25]
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[http://partsregistry.org/Part:BBa_K320008 *C3 + S + Immu + ori + Kan]
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[http://partsregistry.org/Part:BBa_K320009 *C3 + S + P28 + Ori + Tet]
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-
 
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[http://partsregistry.org/Part:BBa_K320011 *C3 + Ori + Amp]
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[http://partsregistry.org/Part:BBa_K320012 *C3 + W + Immu]
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[http://partsregistry.org/Part:BBa_K320013 *C3 + W + P25]
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Here you get a global overview of our BioBricks construction plan.
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<html>
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<img src="https://static.igem.org/mediawiki/2010/f/f4/Three.png" usemap = #third_tree border=0>
 +
<map name=third_tree>
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<area shape=Rect Coords=21,30,86,54 Href="http://partsregistry.org/Part:BBa_K320000">
 +
<area shape=Rect Coords=29,77,85,101 Href="http://partsregistry.org/Part:P1002">
 +
<area shape=Rect Coords=25,120,94,144 Href="http://partsregistry.org/Part:P1003">
 +
<area shape=Rect Coords=25,165,93,189 Href="http://partsregistry.org/Part:P1005">
 +
<area shape=Rect Coords=220,32,346,57 Href="http://partsregistry.org/Part:BBa_K320011">
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<area shape=Rect Coords=224,85,346,113 Href="http://partsregistry.org/Part:BBa_K320004">
 +
<area shape=Rect Coords=224,141,344,169 Href="http://partsregistry.org/Part:BBa_K320003">
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<area shape=Rect Coords=24,282,98,333 Href="http://partsregistry.org/Part:BBa_K320002">
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<area shape=Rect Coords=24,349,97,369 Href="http://partsregistry.org/Part:BBa_K320010">
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<area shape=Rect Coords=24,420,94,450 Href="http://partsregistry.org/Part:BBa_K320001">
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<area shape=Rect Coords=24,470,93,492 Href="http://partsregistry.org/Part:BBa_K320015">
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<area shape=Rect Coords=24,505,86,532 Href="http://partsregistry.org/Part:BBa_K320016">
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<area shape=Rect Coords=25,541,98,572 Href="http://partsregistry.org/Part:BBa_E0020">
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<area shape=Rect Coords=222,292,362,341 Href="http://partsregistry.org/Part:BBa_K320006">
 +
<area shape=Rect Coords=224,372,361,422 Href="http://partsregistry.org/Part:BBa_K320007">
 +
<area shape=Rect Coords=220,508,373,557 Href="http://partsregistry.org/Part:BBa_K320005">
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<area shape=Rect Coords=470,122,725,172 Href="http://partsregistry.org/Part:BBa_K320009">
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<area shape=Rect Coords=469,208,725,261 Href="http://partsregistry.org/Part:BBa_K320008">
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<area shape=Rect Coords=470,366,860,420 Href="http://partsregistry.org/Part:BBa_K320014">
 +
</map>
 +
</html>
 +
== Promoter characterization ==
 +
To characterize the promoter we wanted to use in ''Asaia'' and ''E. coli'' we cloned it in front of a gene coding for CFP. By monitoring the CFP expression over time and comparing to a negative control that did not contain the CFP gene we could measure the activity of the promoter. We also recorded the growth of the two different cultures to see if expression would decrease the growth rate.<br />
 +
Both measurements were done at the same time in ''E. coli'' DH5a over 16 hours at 37°C and the results are averaged over 6 samples.<br />
 +
Unfortunately we can't compare our measurements with a well known promoter.
 +
Further characterization will focus on the measurement of the activity in ''Asaia''.
 +
[[Image:EPFL OD prom.png|800px|center]]
 +
[[Image:EPFL_CFP_prom.png|800px|center]]
{{EPFL_2010_bottom_wrap}}
{{EPFL_2010_bottom_wrap}}
</div>
</div>

Latest revision as of 20:33, 27 October 2010

ork in E. coli, which is very useful for our sets of experiments.



Cloning

caption

We performed all the cloning in E.coli, and ran experiments on Asaia transformation, growth, and antibiotic resistance in parallel.

As the E. coli origin of replication does not work in Asaia, we added an origin that is compatible with Asaia (which was recovered from a GFP-plasmid transformed into the Asaia we received from Italy). Conversely, Asaia's origin of replication works in E. coli, which is very useful as all cloning can be performed using standards developed for E. coli.

BioBricks

caption

All our parts are in the pSB1C3 backbone vector (abbreviate C3).

First the legend:

  • Ori = Origin of replication for Asaia and E. coli
  • S = Strong Promoter
  • W = Weak Promoter
  • Immu = immunotoxin
  • P25 = Pfs25 protein
  • P28 = Pfs28 protein
  • Kan = Resistance to Kanamycin
  • Tet = Resistance to Tetracycline


This are the first and basic constructs we made. Just click on the part to get more information.


These are the BioBricks we obtained by combining the parts from above.


Here you get a global overview of our BioBricks construction plan.


Promoter characterization

To characterize the promoter we wanted to use in Asaia and E. coli we cloned it in front of a gene coding for CFP. By monitoring the CFP expression over time and comparing to a negative control that did not contain the CFP gene we could measure the activity of the promoter. We also recorded the growth of the two different cultures to see if expression would decrease the growth rate.
Both measurements were done at the same time in E. coli DH5a over 16 hours at 37°C and the results are averaged over 6 samples.
Unfortunately we can't compare our measurements with a well known promoter. Further characterization will focus on the measurement of the activity in Asaia.


EPFL OD prom.png
EPFL CFP prom.png

wrap bottom

Retrieved from "http://2010.igem.org/Team:EPF_Lausanne/Project_parts"