Team:UNIPV-Pavia/Notebook/Week1Jun
From 2009.igem.org
(Difference between revisions)
(→June, 3rd) |
(→Week from June 1st, to June 7th, 2009) |
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</td> | </td> | ||
+ | <td> | ||
+ | {| | ||
+ | |[[Image:pv_matteo_resusp.jpg|thumb|300px|left|Matteo resuspending BioBricks from iGEM 2009 plates]] | ||
+ | |} | ||
+ | </td> | ||
+ | </tr> | ||
</table> | </table> | ||
+ | |||
+ | *Transformation of resuspended DNA (2 ul) in TOP10 E. coli, plated transformed bacteria and incubated the plates overnight at 37°C. | ||
+ | |||
+ | *We streaked a plate with a 2008 glycerol stock containing E0240 under the control of a constitutive promoter (we call this construct "01") in order to use it in the fluorescence test at Tecan F200 on June 5th. | ||
== <html><font class="dayw_style">June, 4th</font></html> == | == <html><font class="dayw_style">June, 4th</font></html> == | ||
+ | |||
+ | *All the overnight plates showed colonies. Unfortunately, B0034 and B0032 plates also showed a very small amount of unexpected red colonies...(@_@!?) These two parts had been resuspended from iGEM 2009 plate 1, just like Berkeley constitutive promoters, but it is not clear how they could be contaminated... | ||
+ | |||
+ | *01 plate showed green fluorescence under UV rays, as we expected because GFP was expressed constitutively! | ||
+ | |||
+ | *Anyway, we decided to continue our work and we picked one colony from the following plates: | ||
+ | <table width=100%> | ||
+ | <tr><td> | ||
+ | {|cellpadding="20" | ||
+ | |B0030 | ||
+ | |B0031 | ||
+ | |P0440 (tetR protein generator) | ||
+ | |- | ||
+ | |B0032 | ||
+ | |B0033 | ||
+ | |T9002 (AHL-inducible GFP) | ||
+ | |- | ||
+ | |B0034 | ||
+ | |R0040 (Ptet) | ||
+ | |01 | ||
+ | |- | ||
+ | |J23100 (from +4°C) | ||
+ | |J23101 (from +4°C) | ||
+ | |J23118 (from +4°C) | ||
+ | |} | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | and infected 1 ml of LB + Amp. We incubated the inocula at 37°C, 220 rpm for 5 and 1/2 hours. Of course, we carefully avoided to pick red colonies from B0034 and B0032 plates:) Sequencing checks will tell us if our parts are correct. | ||
+ | |||
+ | *Glycerol stocks for all the grown cultures (except for 01, because we already had it). | ||
+ | |||
+ | *NOTE: we re-picked colonies from J23100, J23101 and J23118 plates because data analysis of experiment 1 at Tecan F200 showed that J23101 promoter seemed stronger than J23100. This is not in accordance to the ranking of the promoters (http://partsregistry.org/Promoters/Catalog/Anderson), so we decided to repeat the test at Tecan F200 and to store these now glycerol stocks at -80°C. | ||
+ | |||
+ | *We prepared 5 ml cultures for: | ||
+ | **B0030 (for sequencing because it is the first RBS we wanted to use) | ||
+ | **J23100 (for sequencing because we wanted to check the integrity of the downstream RFP) | ||
+ | **J23100 (for tomorrow's test - RFP assay) | ||
+ | **J23101 (for tomorrow's test - RFP assay) | ||
+ | **J23118 (for tomorrow's test - RFP assay) | ||
+ | **E0240 (for tomorrow's test - negative control) | ||
+ | **T9002 (NOT INDUCED for tomorrow's test - GFP assay to detect leaky activity caused by Plux) | ||
+ | **T9002 (TO INDUCE TOMORROW for the test - GFP assay) | ||
+ | **01 (for tomorrow's test - positive control) | ||
+ | |||
+ | *and incubated them overnight at 37°C, 220 rpm. | ||
== <html><font class="dayw_style">June, 5th</font></html> == | == <html><font class="dayw_style">June, 5th</font></html> == |
Revision as of 13:53, 20 June 2009
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Week from June 1st, to June 7th, 2009
June, 3rd
- We received lactose monohydrate, M9 minimal salts and Thiamine Hydrochloride from Sigma.
- DNA resuspension from iGEM 2009 plates. We resuspended the two parts we needed to re-built the inconsistent BioBrick Q04400, the five commonly used RBSs and a 3OC6-HSL inducible measurement system for GFP test at Tecan F200:
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|
- Transformation of resuspended DNA (2 ul) in TOP10 E. coli, plated transformed bacteria and incubated the plates overnight at 37°C.
- We streaked a plate with a 2008 glycerol stock containing E0240 under the control of a constitutive promoter (we call this construct "01") in order to use it in the fluorescence test at Tecan F200 on June 5th.
June, 4th
- All the overnight plates showed colonies. Unfortunately, B0034 and B0032 plates also showed a very small amount of unexpected red colonies...(@_@!?) These two parts had been resuspended from iGEM 2009 plate 1, just like Berkeley constitutive promoters, but it is not clear how they could be contaminated...
- 01 plate showed green fluorescence under UV rays, as we expected because GFP was expressed constitutively!
- Anyway, we decided to continue our work and we picked one colony from the following plates:
|
and infected 1 ml of LB + Amp. We incubated the inocula at 37°C, 220 rpm for 5 and 1/2 hours. Of course, we carefully avoided to pick red colonies from B0034 and B0032 plates:) Sequencing checks will tell us if our parts are correct.
- Glycerol stocks for all the grown cultures (except for 01, because we already had it).
- NOTE: we re-picked colonies from J23100, J23101 and J23118 plates because data analysis of experiment 1 at Tecan F200 showed that J23101 promoter seemed stronger than J23100. This is not in accordance to the ranking of the promoters (http://partsregistry.org/Promoters/Catalog/Anderson), so we decided to repeat the test at Tecan F200 and to store these now glycerol stocks at -80°C.
- We prepared 5 ml cultures for:
- B0030 (for sequencing because it is the first RBS we wanted to use)
- J23100 (for sequencing because we wanted to check the integrity of the downstream RFP)
- J23100 (for tomorrow's test - RFP assay)
- J23101 (for tomorrow's test - RFP assay)
- J23118 (for tomorrow's test - RFP assay)
- E0240 (for tomorrow's test - negative control)
- T9002 (NOT INDUCED for tomorrow's test - GFP assay to detect leaky activity caused by Plux)
- T9002 (TO INDUCE TOMORROW for the test - GFP assay)
- 01 (for tomorrow's test - positive control)
- and incubated them overnight at 37°C, 220 rpm.
June, 5th
Experiment with Tecan F200
- Description
- Purpose:
- Materials & Methods
- Protocol
- Results
June, 6th
Experiment with Tecan F200
- Results check