Team:UNIPV-Pavia/Notebook/Week1Jun

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__NOTOC__  
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= Week from June 1st, to June 7th, 2009 =
= Week from June 1st, to June 7th, 2009 =
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<td align="left" width="50%">
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<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5May">
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<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5May#week_start">
  <img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/>
  <img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/>
</a>
</a>
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<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5May">Previous Week</a>
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<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5May#week_start">Previous Week</a>
</td>
</td>
<td align="right" width="50%">  
<td align="right" width="50%">  
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<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Jun">Next Week</a>
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<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Jun#week_start">Next Week</a>
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<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Jun">
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<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Jun#week_start">
  <img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/>
  <img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/>
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{|
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<font class='didascalia'>
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|[[Image:pv_matteo_resusp.jpg|thumb|300px|left|Matteo and Lorenzo resuspending BioBricks from iGEM 2009 plates]]
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[[Image:pv_matteo_resusp.jpg|thumb|300px|left|Matteo and Lorenzo resuspending BioBricks from iGEM 2009 plates]]
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**J23101 (for tomorrow's test - RFP assay)
**J23101 (for tomorrow's test - RFP assay)
**J23118 (for tomorrow's test - RFP assay)
**J23118 (for tomorrow's test - RFP assay)
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**E0240 (for tomorrow's test - negative control)
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**E0240 (for tomorrow's test - negative control, picked from the plate stored at +4°C)
**T9002 (NOT INDUCED for tomorrow's test - GFP assay to detect leaky activity caused by Plux - we will call it T9002-)
**T9002 (NOT INDUCED for tomorrow's test - GFP assay to detect leaky activity caused by Plux - we will call it T9002-)
**T9002 (TO INDUCE TOMORROW for the test - GFP assay - we will call it T9002+)
**T9002 (TO INDUCE TOMORROW for the test - GFP assay - we will call it T9002+)
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{|
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<font class='didascalia'>
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|[[Image:pv_susanna_colonypcr.jpg|thumb|220px|left|Susanna preparing samples for colony PCR]]
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[[Image:pv_susanna_colonypcr.jpg|thumb|220px|left|Susanna preparing samples for colony PCR]]
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<table align="center">
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|[[Image:pv_pcr_p0040_r0040.jpg|thumb|300px|left|Colony PCR: P0040 insert was amplified correctly, while R0040 showed contaminants or unexpected bands]]
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*Gel results: the 3 colonies of P0440 all have the plasmid with the correct length of the insert (1078 bp), while there have been problems with R0040 colonies. R0040-1 and R0040-2 show the correct length of the insert (292 bp) with a high weight contaminant and R0040-3 show an unexpected wrong length. Next week we are going to digest P0440 and R0040 to perform their assembly, so we will check the actual plasmid and insert length again.
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<font class='didascalia'>
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[[Image:pv_pcr_p0040_r0040.jpg|thumb|300px|left|Colony PCR: P0040 insert was amplified correctly, while R0040 showed contaminants or unexpected bands]]
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*Gel results: the 3 colonies of P0440 all have the plasmid with the correct length of the insert (1078 bp), while there have been problems with R0040 colonies. R0040-1 and R0040-2 show the correct length of the insert (292 bp) with a high weight contaminant and R0040-3 show an unexpected wrong length. Next week we are going to digest P0440 and R0040 to perform their assembly, so we will check the actual plasmid and insert length again.
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{|
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|[[Image:pv_mini_redyellow.jpg|thumb|230px|left|Red pellet for J23100-seq and normal-coloured pellet for B0030 after 7 min at 7000 rpm centrifuge]]
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[[Image:pv_mini_redyellow.jpg|thumb|230px|left|Red pellet for J23100-seq and normal-coloured pellet for B0030 after 7 min at 7000 rpm centrifuge]]
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<font class='didascalia'>
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|[[Image:pv_ahl_vial.jpg|thumb|150px|left|3OC6-HSL 2 mM vial]]
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[[Image:pv_ahl_vial.jpg|thumb|150px|left|3OC6-HSL 2 mM vial]]
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''Experiment with Tecan F200''
''Experiment with Tecan F200''
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* <html><u>Description</u></html>
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*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/J100 dil e T9002 ind TEST 05-06-09.pdf" target="_blank">Download Protocol</a></html>
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* <html><u>Purpose:</u></html>
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* <html><u>Materials & Methods</u></html>
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* <html><u>Protocol</u></html>
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* <html><u>Results</u></html>
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<tr>
<tr>
<td align="left" width="50%">
<td align="left" width="50%">
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<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5May">
+
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5May#week_start">
  <img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/>
  <img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/>
</a>
</a>
-
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5May">Previous Week</a>
+
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week5May#week_start">Previous Week</a>
</td>
</td>
<td align="right" width="50%">  
<td align="right" width="50%">  
-
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Jun">Next Week</a>
+
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Jun#week_start">Next Week</a>
-
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Jun">
+
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week2Jun#week_start">
  <img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/>
  <img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/>
</a>
</a>

Latest revision as of 14:14, 20 October 2009

EthanolPVanimation.gif

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Week from June 1st, to June 7th, 2009

Previous Week Next Week

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:
B0030 B0031 P0440 (tetR protein generator)
B0032 B0033 T9002 (AHL-inducible GFP)
B0034 R0040 (Ptet)

Matteo and Lorenzo resuspending BioBricks from iGEM 2009 plates
  • 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.

Top

June, 4th

  • All the overnight plates showed colonies. 01 plate showed green fluorescence under UV rays, as we expected because GFP was expressed constitutively!

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...

  • Anyway, we decided to continue our work and we picked one colony from the following plates:
B0030 B0031 P0440 (tetR protein generator)
B0032 B0033 T9002 (AHL-inducible GFP)
B0034 R0040 (Ptet) 01
J23100 (from +4°C plate) J23101 (from +4°C plate) J23118 (from +4°C plate)

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 with brand new colonies and to store these new glycerol stocks at -80°C.
  • We prepared 5 ml cultures (re-filling the remaining 250 ul of the grown bacteria) for:
    • B0030-seq (for sequencing because it is the first RBS we wanted to use)
    • J23100-seq (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, picked from the plate stored at +4°C)
    • T9002 (NOT INDUCED for tomorrow's test - GFP assay to detect leaky activity caused by Plux - we will call it T9002-)
    • T9002 (TO INDUCE TOMORROW for the test - GFP assay - we will call it T9002+)
    • 01 (for tomorrow's test - positive control)
  • and incubated them overnight at 37°C, 220 rpm.
  • Colony PCR for P0440(3 colonies) and R0040(3 colonies) plates to check for contaminations.
  • Gel run for the resulting reactions.

Susanna preparing samples for colony PCR

Colony PCR: P0040 insert was amplified correctly, while R0040 showed contaminants or unexpected bands
  • Gel results: the 3 colonies of P0440 all have the plasmid with the correct length of the insert (1078 bp), while there have been problems with R0040 colonies. R0040-1 and R0040-2 show the correct length of the insert (292 bp) with a high weight contaminant and R0040-3 show an unexpected wrong length. Next week we are going to digest P0440 and R0040 to perform their assembly, so we will check the actual plasmid and insert length again.


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June, 5th

  • All the overnight cultures were grown. RFP-expressing cultures were red-coloured, as usual!
  • Miniprep for B0030-seq and J23100-seq.
  • We sent B0030 and J23100 to BMR Genomics for sequencing.

Preparation of the second experiment with Tecan F200

  • We diluted 1:10 these overnight cultures:
    • J23100
    • J23101
    • J23118
    • E0240
    • T9002-
    • T9002+
    • 01
  • We induced the diluted and non-diluted cultures of T9002+ with 3OC6-HSL 200 nM.
  • We incubated all the diluted and non-diluted cultures at 37°C, 220 rpm for 4 and 1/2 hours.

Red pellet for J23100-seq and normal-coloured pellet for B0030 after 7 min at 7000 rpm centrifuge

3OC6-HSL 2 mM vial

Experiment with Tecan F200

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June, 6th

Experiment with Tecan F200

  • Results check



Top

Previous Week Next Week

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