Calgary/design
From 2007.igem.org
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| Projects | Design: Wet Lab | Design: Printer | Design: Software | Testing | Construction: The Wetlab | Protocols | Final Result of E.co Lisa |
Welcome to our Teams wetalab section. Our wetlab entry consists of several different components, which are described in this section. Just click on the compenent you want to learn more about
Logic Circut |
Agarase |
BioMarkers |
Light Sensor |
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The sucess of our project is dependent on us having very percise control over the expression of our reporter gene. In order to draw a legible picture our system needs to be able to induce very high expression of agarase under the desired conditions and then almost completely stop agarase expression when the activating conditions are removed. This is because an over expression of agarase might just degarde the entire plate leaving nothing visible as a picture. Initially one of our team members, Dave Curran, designed a very slick and complex system to regulate expression. However as we were running out of time we were forced to rely on a more simplified version. The simplified version works as follows.
In the absence of 660nm light and AHL, both promoters in the circuit are repressed. If the bacteria were then to be exposed to light, the protein LuxR would be produced, but would not activate the promoter lux pR. When AHL is added to the cells that are still in the dark, the lux pR promoter would still not be activated, as the protein LuxR would not be present. But if AHL is added to the cells, and they are then exposed to light, both AHL and LuxR will be present, and so the lux pR promoter will be activated. When this occurs the reporter gene is expressed, and more LuxR is produced so that the system will remain on even when the light is then taken away.
The complicated system functions in much the same way, except it contains one additional level of security, to prevent accidental activation of the system. There is an RNA lock covering the ribosome binding site in front of the second luxR in the plasmid. This way, even if the lux pR promoter is a touch leaky, no LuxR will be produced to fully activate the system.
The application below shows the schematics of both the complex and simple systems. Hovering over a part with the mouse will highlight its corresponding description in the table. Clicking on a part in the diagram will open the registry's page that desribes the part.
NOTE: if you are viewing this page with internet explorer you will have to click on the application once before you can use it
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In addition to our logic control circut our team has also done significant work with the protein β agarase. β agarase is a protein native the marine bacterial species Pseudoalteromonas atlantica . In its natural environment this bacteria uses β agarase to degrade agar like components of seaweed into nutrient sugars. We have attempted to adapt this gene to be expressed in E. coli in such a way that its expression will activated by our light sensing system (for more on the light sensor see the light sensor section of this page). The light sensor activates the logic circut described above which will initiate highly controlled expression of agarase.
Using the primers we designed we were able to successfully isolate the β agarase gene from the Pseudoalteromonas atlantica genome and place it into a BioBrick plasmid (sequencing information for β agarase is available in our final results section). However we were unable to express β agarase in E. coli. Our current results indicate that while β agarase is being produced, our E. coli cells are unable to export the protein.
Pseudoalteromonas atlantica β-agarase DNA sequence
- RED - start codon
- BLUE - stop codon
- GREEN - forward primer annealing region
- PINK- reverse primer annealing region
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T G C A A A A C A C C A G C C C A G A T T C A G T T C A T T T G T A C T T G T C T T T G G A A A A T G T A A A C G G A C A A A T C C A G A G T C A C T C A A T T T C T C T G G C T C C G A A T T A C A A A G G C A C G G T G T A T T T T C C C C T C G A T G G T A T T G A A G C A G A A A C C G A C A G C G G C A T G T G G G G A G A T G T G C C T C A T T G G C A A A C C C A A G A C G A C T T A A T G G T G T G G C G T T C T T G G C G C A A A G C A G A G C A A G A C T A T A G C C A A C T C A C C G C G C T T A A T T T C T T C A C T A T T G G A A T A T T G G A A A A C A A A T C T G T G A T T T T G G A T G A T G T G C G T C T G C G C G C G A A T C C A G C T C A C G A C C C G A A C G T A T C G G T C G G A T T A A T C G A T A A A T A C G G G C A A A A C G C C A A A C A A A G C A C C C C C T T A G A T G T G C A T T C A G A T G C C C A A C T A A A A C A A C A A G C C G A T G A A G A A C T G G C C A A G C T A G C C A A A T C A T C G G G T A T G C C A G A C C G T T C T C G T T T T G G T G G T T A T A C C A A A G C C C C A A A A C G T G A A G C A A C C G G C T T T T T T A G A A C G G A A A A G G T C G A T G G T A A G T G G T G G A T G G T C G A C C C T G A T G G C T A T C T G T T T T T C T C A C A C G G G C C A G C T A A C G T G C G T A T G G C A A A C A T G A G T A C G T T A A C C G G A A T A G A C T A C G A C C A A C C G A G T A T T C G C G T G C G C A C C A G T G A T G A A A T T A C C C C A G A A G A T T C C A T G G G A A T T G T A T C T G T T C C T G A C A G C G C A A A A G A A A A A C G C T A T G T C A T T T C A A A G G C G C G C A A C G A T A T G T T T C A G T G G T T A C C A A G C T A T G A T G A T G A G C T C T C T G A G C A C T A T A G C T A T C G C C G C T C T A C C C A C A A A G G G C C T A T T C C T T A C G G C G A G A C C T A C A G C T T T T A C C G C G C T A A T T T A G A A C G T A G A T A C G G T G A T G A C G A C C T A C G C G C A G G T G A A G G T G A A A A A G C A G G C G A A A A T C A A G G C A G C G A T C C T T C C T A C G T A A A A A A A T G G C A T G A C G T C A C C G C A C A G C G T A T G C A T G A C T G G G G G T T T A C C T C T T T T G G T A A C T G G G T A G A C C C A G C G T T T T A T C A A T C A G A G C A A G T G C C T T A T T T C G C C A A T G G T T G G A T C A T C G G C G A T T A C C A A A C A C T G T C G G G T C A C A C T A A T C A T T G G G G C T T G A T G C C A G A T C C T T T T G A T C C C G T A T T T G C A C A G C G A G C A A A G G T C A C T A T C G A T G C T A T T G C C G A A A A T A T T C A A G C G T C A C C T T G G T G T G C G G G T A T T T T T A T T G A T A A T G A G A A G A G C T G G G G A G A G C G T G A A G G C A C A G T G T C A C A A C G C T A T G G G G T G A T C C T T G A T G C A C T A T C A A A A A A T A T T G A G A A G A G C C C T G C A A A A A C G G C C T T T G C A G C C C A T T T G C A G C A A A A A T A C C A G A C G A T C G A T G C A T T G A A T T C T G C A T G G T C A T C A C A C T T C G A T A G T T G G C A A A T G T T T G A A G A T G A C G C C A G C G T C A G C A C G C A C A C C G A T G C C C A A A T T G C T G A T T T G T C A A A A A T G C T C G A G A T G C T G G G T G A G C A G T A C T T T A A A G T G G T A C A C A A T A C C T T A G A A G C G G C G C T G C C A G A G C A C T T G T A C A T G G G C G C G C G C A T G G C C A A C T G G G G C A T G C C T G A T G A A A T C A T T A C T G C C T C T T T G A A A T A C T C T G A C G T A C T G A G C T T C A A T A T T T A T G A G G A A G G G G T G C A A G A A G A C T A T T G G A A G T T C C T T G A A G A A G T C G A C T T A C C C G T A G T G A T T G G T G A G T T T C A T A T T G G C A G C A C G A C A G A T T C A G G C A T G T A C A A C C C G G G T A T T G T G C A C A G C G C A A A C C A G C A A G A C A G A G C C C A A A T G T A C A A A G A T T A C A T G C A A A G C G T G T T A G A C A A G C C G T A T A T G G T C G G G G C C C A T T G G T T T C A A T A C A T A G A T G A G C C C A T A T C G G G G C G T G C A T T T G A C G G C G A A A A C G C C A A T A T C G G C T T T G T A A C G G T A A C G G A T A T C C C A T A T C C G C A T A T G A T C C A A G C G G T C A A A G A G G T G A C A T C C A C C A T G T A C C A A A A A C G C T T A G C C G A C T A A T A C A G G C T T A G T G A T A A C A A A A A A C G C C C G A T A A C G T C G G G C G T T T T G C G T T T G T A A A C G A C T A T
Pseudoalteromonas atlantica β-agarase amino acid sequence
M N T S P K S A G F I L S A I A L A V T A L A G C N N N Q A T N N S V A T P S D A P T R Q T Q Q N V L P W D F A G V T D M Q D V T L T A A D M H V V D V D G E Q K L A I D L H A K E H K S A G F S F I P D S P W D W S H E G Q F A F A I E I E N P S Q S S T H L Y V S V K D A T G Q S H N R S F A V P G H S Q D T Y F M A L N D P D L S I E T G I R S N P N N W Q S E F T P M I W R Y G T K K I D L S Q V K S I E F D V R G V P E D K H L I V D N P R L I K P Q K I D Q N Y L V G L V D E F G Q N D K L E F T N K I D T V E Q L R A L N A K E Q S A F T H D V P K G R S K F N G W A D G P K L A A T G Y F R T E K Y Q G K W T L V D P Q G Y L F F S N G I A N V R M S N T S T I T G Y D F D S Q F I K Q R A Q G D F T P E D S I G L N R A P K A A W P S R H V T S E L R A N M F T W L P S V D E P L A E H F G Y R R E V H T G V V D K G E T Y S F Y S A N L A R K Y A S N D P K V F M P K W R D T T V D R M L D W G F T S F G N W I D P S F Y Q L N R I P Y F A N G W I I G D Y K T V S S G N D Y W S P L P D P F D P V F K Q R A M V T A N K I A Q E V Q N N P W C V G V F I D N E K S W G Q E G S I E R Q Y G I A I H T L E V D A K D S P T K A Q F V T Y L K N K Y S D I A E L N G K W N T Q I R T W D D V A T G I T L T R F N D P I I E D L S A M L S L Y A E K Y F A V V H D A V E Q A M P N H M Y M G V R F A D W G M T D E I R N A A A K Y A D V V S Y N Y Y K E A I T D Q A W G F L A K I D K P S I I G E F H N G A L D S G L L N P G L I H S A S Q A D R G K K Y Q E Y I N S V I D N P Y F V G A H W F Q Y I D S P L T G R A Y D G E N Y N V G F V S V T D T P Y Q P L V D A A K A V N A N I Y T R R F G D A Q P K
Pseudoalteromonas atlantica β-agarase Primers
Oligo |
Start |
Length |
Melting Temp |
GC% |
Any |
3' Sequence |
Left Primer |
28 |
20 |
60.30 C |
55.00 |
8.00 |
T A G G C T C T G C T C A G C C T T G T |
Right Primer |
2639 |
19 |
59.87 C |
57.89% |
4.00 |
C G C G G T C G A G T G G T A G T T A |
Effect of agarase on agar
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Outside of the laser interface our team experimented with diffusable signalling molecules as another way of developing a highly controlled human-bacteria interface. The goal of the BioMarker project was to use an AHL induced circut to produce images on E. coli. Ideally AHL could be selectively applied to specific parts of a plate containing our engineered cells. The cells exposed to AHL would then express our reporter gene, GFP. This would allow us to "draw" with AHL on the agar and the bacteria would then glow in the patter that was drawn. This project made use of the construct from the U of C 2006 iGEM project, F2620+L13504.
BioMarker in action
the bacteria are pleased
There were two approaches considered for this project. The first was to have an inkjet mounted on our laser plotter that would apply AHL to the agar. The second was to use a "BioMarker". That is a marker infused with AHL solution. While we have selected the latter for our project, the printer system remains a viable option that may be considered in the future. The BioMarkers were made using Fantastix blank markers by Tsukineko Corporation. The AHL was dissolved in acidified ethyl acetate to a 1uM concentration and then moved into the markers using a transfer pipette. The non-polar core of the Fantastix blank is well suited to absorbing the ethyl acetate dissolved AHL.
Early tests of the BioMarkers on regular agar plates have yielded promising results. While the agar does not readily absorb the ethyl acetate, it absorbs enough to get expression in a 3mm path around the line drawn with the thin tip type of BioMarker.
Our original plan called for applying a layer of top agar, that is Agar that is less dense than regular agar, over the normal agar containing our cells. However our experiements with Top Agar have shown that Top Agar is too soft to be draw in. After a uniform lawn has been grown overnight, the 1uM BioMarker is used to draw on the agar. The response is seen after 2 hours of incubation at 37 C.
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