IGEM:Paris Bettencourt 2012/Notebooks/modularity group
Purpuse of this group
Projects
PKU 2010 [1]
concept
the circuit
comment on the modularity
Cambridge 2010 [2]
concept
the circuit
comment on the modularity
Bristol 2010 [3]
encapsulation technique:[ http://www.freepatentsonline.com/5093253.html]
concept
the circuit
more details in the future
Characterization by Bristol 2010 Team
Two 5mL overnights were set up, one without any nitrogen and one with 20mM of Potassium Nitrate KNO3, made up according to the table below. This level was chosen as previous work by the Edinburgh 2009 iGEM team had shown the promoter strongly active by this point.
| Nitrate (mM) | LB (mL) | KNO3 (μL) |
|---|---|---|
| 0 | 5 | 0 |
| 20 | 5 | 102 |
The cultures were then left in the 37°C shaker overnight and examined the next morning. We were pleased to note that the culture exposed to nitrate was visibly green whilst the one without was not, suggesting our new BioBrick worked at least at the most basic ‘on/ off’ level.
Our next step was to set up and assay examining the BioBrick's behaviour between 0 – 10 mM of Potassium Nitrate at 2mM intervals. The same colony of MG1655 containing BBa_K381001 was used to inoculate six 5mL tubes of LB at varying nitrate levels as listed below. This was repeated twice to allow the results to be averaged.
| Nitrate (mM) | LB (mL) | KNO3 (μL) | GFP level test 1 | GFP level test 2 | Average GFP level |
| 0 | 5 | 0 | 0.0242 | 0.0289 | 0.0266 |
| 2 | 5 | 10 | 0.0752 | 0.1143 | 0.1948 |
| 4 | 5 | 20 | 0.1042 | 0.1197 | 0.1120 |
| 6 | 5 | 30 | 0.1248 | 0.1234 | 0.1241 |
| 8 | 5 | 40 | 0.1179 | 0.1396 | 0.1288 |
| 10 | 5 | 50 | 0.0631 | 0.1186 | 0.0909 |
Materials
E coli strains
DH5α Turbo (NEB): the cells that we have.
obtained from ...... fridge. (day and link)
F´ proA+B+ lacIq ∆ lacZ M15/ fhuA2 ∆(lac-proAB) glnV gal R(zgb-210::Tn10)TetS endA1 thi-1 ∆(hsdS-mcrB)5 Also known as NEB Turbo
T1 phage resistant Rapid growth: visible colonies on agar, ~6.5 hours; shaking liquid culture OD 600 = 2.0, ~4 hours Expresses the Lac repressor References: New England Biolabs, product catalogue number C2984H
top10 strain
received from the ...... the ...th of July. (link to day by day notebook)
Subtilis strains
BSPY79: wild type. SB513: Kan resistant. SB473: CM resistant.
comment on the modularity
What we are doing now
PKU 2010
communication, requests of plasmids. searching for mercury, it seems that we need 2 strains: BL21DE3 and DH5 alpha depending of the part of the project we want to do. (both?)
Cambridge 2010
We have the biobrick we want to use (but there are 2 in fact): 2012 Kit Plate 4 well 17M, resistance: C But should we go on with that project? it uses the pBad promoter.
Bristol 2010
We have the biobrick: complete system: 2012 Kit Plate 4 well 1A, resistance: C
Nitrate needed: Potassium Nitrate
Characterization by Bristol 2010 Team: Two 5mL overnights were set up, one without any nitrogen and one with 20mM of Potassium Nitrate KNO3, made up according to the table below. This level was chosen as previous work by the Edinburgh 2009 iGEM team had shown the promoter strongly active by this point.
| Nitrate (mM) | LB (mL) | KNO3 (μL) |
|---|---|---|
| 0 | 5 | 0 |
| 20 | 5 | 102 |
The cultures were then left in the 37°C shaker overnight and examined the next morning. We were pleased to note that the culture exposed to nitrate was visibly green whilst the one without was not, suggesting our new BioBrick worked at least at the most basic ‘on/ off’ level.
Our next step was to set up and assay examining the BioBrick's behaviour between 0 – 10 mM of Potassium Nitrate at 2mM intervals. The same colony of MG1655 containing BBa_K381001 was used to inoculate six 5mL tubes of LB at varying nitrate levels as listed below. This was repeated twice to allow the results to be averaged.
| Nitrate (mM) | LB (mL) | KNO3 (μL) | GFP level test 1 | GFP level test 2 | Average GFP level |
| 0 | 5 | 0 | 0.0242 | 0.0289 | 0.0266 |
| 2 | 5 | 10 | 0.0752 | 0.1143 | 0.1948 |
| 4 | 5 | 20 | 0.1042 | 0.1197 | 0.1120 |
| 6 | 5 | 30 | 0.1248 | 0.1234 | 0.1241 |
| 8 | 5 | 40 | 0.1179 | 0.1396 | 0.1288 |
| 10 | 5 | 50 | 0.0631 | 0.1186 | 0.0909 |
References
gene tranfert experiment design
naturally competent bacteria : chen-dubnau-2004 johnsborg-etal-2007 E coli antibiotic in subtilis: http://opus.bibliothek.uni-wuerzburg.de/volltexte/2011/6060/pdf/Kreft13.pdf
