Team:Macquarie Australia/Protocols/Outreach Planning

From 2012.igem.org

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For outreach we wanted to make colourful or glowing E. Coli.
For outreach we wanted to make colourful or glowing E. Coli.
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|+ align="centre" style="color:#e76700;" |
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!Biobrick Name
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!Registry Part
!Plasmid
!Plasmid
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!Plate
 
!Well
!Well
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!Plate
!Promoter
!Promoter
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!Characteristic(s)
!Gene
!Gene
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|BBa_I13521
|BBa_I13521
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|pSB1A3 2
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|pSB1A3  
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|60
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|6O
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|2
|TetR
|TetR
|repressible
|repressible
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|BBa_I13522
|BBa_I13522
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|pSB1A2 2
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|pSB1A2  
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|A8
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|8A
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|2
|TetR
|TetR
|repressible
|repressible
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|BBa_I13600
|BBa_I13600
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|pSB1A2 1
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|pSB1A2  
|24E
|24E
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|1
|TetR  
|TetR  
|repressible
|repressible
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|BBa_I712052
|BBa_I712052
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|pSB1AK8 2
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|pSB1AK8  
|13G
|13G
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|T&
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|2
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|T7
|bacteriophage
|bacteriophage
|Luciferase
|Luciferase
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|BBa_K274210
|BBa_K274210
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|pSB1A2 3
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|pSB1A2  
|6N
|6N
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|3
|TetR
|TetR
|repressible
|repressible
|Orange Fluorescent Protein (OFP)
|Orange Fluorescent Protein (OFP)
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We chose five different genes from the IGEM parts registry that were fluorescent or bio-luminescent (as shown in the table above): red, blue, orange and green fluorescent proteins, as well as luciferase. The fluorescent proteins were all transformed into Top 10 competent E-coli as they have TetR repressible promoters, while Luciferase it was transformed into BL21-De3 (Origami) competent E. coli cells as it has a T7 promoter. All competent cells were obtained from Robert Willows lab.
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We chose five different genes from the IGEM parts registry that were fluorescent or bio-luminescent (as shown in the table above): red, blue, orange and green fluorescent proteins, as well as luciferase. The fluorescent proteins were all transformed into Top 10 competent E-coli as they have TetR repressible promoters, while Luciferase it was transformed into BL21-DE3 (Origami) competent E. coli cells as it has a T7 promoter. All competent cells were obtained from Robert Willows lab.
 +
[[File:Photo1.jpg|200px|thumb|left|iGEM plates from which parts were obtained]]
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The Top 10 E. coli cells were divided into 4 x 50 μl aliquots, while 100 μl of BL21 was measured out.  We located the corresponding wells for the gene plasmids in the plates supplied in our parts kit, and resuspended each plasmid in 10 μl of MilliQ H20. We then added 1μl of each plasmid to the relevant E.coli cells for transformation. We incubated the cells for 5 mins on ice, subjected them to heat shock at 42o C, and placed them on ice for a further 2 mins. We then added 500 μl of SOC media to each tube and incubated them for an hour at 37o C in a shaker.  
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The Top 10 E. coli cells were divided into 4 x 50 μl aliquots, while 100 μl of BL21 was measured out.  We located the corresponding wells for the gene plasmids in the plates supplied in our parts kit, and resuspended each plasmid in 10 μl of MilliQ H20. We then added 1μl of each plasmid to the relevant E.coli cells for transformation. We incubated the cells for 5 mins on ice, subjected them to heat shock at 42&deg;C, and placed them on ice for a further 2 mins. We then added 500 μl of SOC media to each tube and incubated them for an hour at 37&deg;C in a shaker.
 +
1mL of 0.1M IPTG stock solution was added to 9mL of MilliQ H2O giving a final concentration of 0.01M.  This final IPTG was then used for the plates. 
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Each of the transformations were then plated out on ampicillin plates as follows:  
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Each of the transformations were then plated out on ampicillan plates as follows:  
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1 x 20 μl on plates with added IPTG<br>
1 x 20 μl on plates with added IPTG<br>
1 x 200 μl on plates with added IPTG<br>
1 x 200 μl on plates with added IPTG<br>
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1 x 200 μl on plates without IPTG<br>
1 x 200 μl on plates without IPTG<br>
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The plates were then incubated overnight at 37o C.
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The plates were then incubated overnight at 37&deg;C.
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* Due to using 20 ampicillin plates from our stock of plates for use with our project, we made up 20 more LB agar plates on this day as well.

Latest revision as of 02:37, 27 September 2012



For outreach we wanted to make colourful or glowing E. Coli.

Registry Part Plasmid Well Plate Promoter Characteristic(s) Gene
BBa_I13521 pSB1A3 6O 2 TetR repressible Red Fluorescent Protein (RFP)
BBa_I13522 pSB1A2 8A 2 TetR repressible Green Fluorescent Protein (GFP)
BBa_I13600 pSB1A2 24E 1 TetR repressible Cyan Fluorescent Protein (CFP)
BBa_I712052 pSB1AK8 13G 2 T7 bacteriophage Luciferase
BBa_K274210 pSB1A2 6N 3 TetR repressible Orange Fluorescent Protein (OFP)


We chose five different genes from the IGEM parts registry that were fluorescent or bio-luminescent (as shown in the table above): red, blue, orange and green fluorescent proteins, as well as luciferase. The fluorescent proteins were all transformed into Top 10 competent E-coli as they have TetR repressible promoters, while Luciferase it was transformed into BL21-DE3 (Origami) competent E. coli cells as it has a T7 promoter. All competent cells were obtained from Robert Willows lab.

iGEM plates from which parts were obtained

The Top 10 E. coli cells were divided into 4 x 50 μl aliquots, while 100 μl of BL21 was measured out. We located the corresponding wells for the gene plasmids in the plates supplied in our parts kit, and resuspended each plasmid in 10 μl of MilliQ H20. We then added 1μl of each plasmid to the relevant E.coli cells for transformation. We incubated the cells for 5 mins on ice, subjected them to heat shock at 42°C, and placed them on ice for a further 2 mins. We then added 500 μl of SOC media to each tube and incubated them for an hour at 37°C in a shaker.

1mL of 0.1M IPTG stock solution was added to 9mL of MilliQ H2O giving a final concentration of 0.01M. This final IPTG was then used for the plates.

Each of the transformations were then plated out on ampicillin plates as follows: 1 x 20 μl on plates with added IPTG
1 x 200 μl on plates with added IPTG
1 x 20 μl on plates without IPTG
1 x 200 μl on plates without IPTG

The plates were then incubated overnight at 37°C.

  • Due to using 20 ampicillin plates from our stock of plates for use with our project, we made up 20 more LB agar plates on this day as well.
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