Team:UNIPV-Pavia/Notebook/Week5May

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= Week from May 25th, to May 31st, 2009 =
= Week from May 25th, to May 31st, 2009 =
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<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4May#week_start">
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<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/>
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</a>
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<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4May#week_start">Previous Week</a>
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</td>
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<td align="right" width="50%">
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<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Jun#week_start">Next Week</a>
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<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Jun#week_start">
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<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/>
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</a>
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</td>
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== <html><font class="dayw_style">May, 25th</font></html> ==
== <html><font class="dayw_style">May, 25th</font></html> ==
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*We planned to dedicate the first two weeks to prepare a glycerol stock for every BioBrick of interest for our project. The following weeks will be dedicated to the BioBrick assembly.
*We planned to dedicate the first two weeks to prepare a glycerol stock for every BioBrick of interest for our project. The following weeks will be dedicated to the BioBrick assembly.
 +
*DNA resuspension from iGEM 2009 plates. We resuspended all the high quality constitutive promoter family members from UCBerkeley collection and GFP protein generator:
*DNA resuspension from iGEM 2009 plates. We resuspended all the high quality constitutive promoter family members from UCBerkeley collection and GFP protein generator:
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|E0240
|E0240
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|}
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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.
*Transformation of resuspended DNA (2 ul) in TOP10 E. coli, plated transformed bacteria and incubated the plates overnight at 37°C.
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[[#top|Top]]
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== <html><font class="dayw_style">May, 26th</font></html> ==
== <html><font class="dayw_style">May, 26th</font></html> ==
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<tr valign="up">
<td>
<td>
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{|
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<font class='didascalia'>
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|[[Image:pv_firstday_plates.jpg|thumb|300px|left|High quality Berkeley promoters and E0240 plates: high yield for all of them!]]
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[[Image:pv_firstday_plates.jpg|thumb|300px|left|High quality Berkeley promoters and E0240 plates: high yield for all of them!]]
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|}
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</font>
</td>
</td>
<td>
<td>
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{|
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<font class='didascalia'>
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|[[Image:pv_j102_plate.jpg|thumb|300px|left|J23102 plate: the promoter is very strong and RFP expression could be seen in the red-coloured colonies. The same result was seen in the other strong Berkeley promoters.]]
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[[Image:pv_j102_plate.jpg|thumb|300px|left|J23102 plate: the promoter is very strong and RFP expression could be seen in the red-coloured colonies. The same result was seen in the other strong Berkeley promoters.]]
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|}
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</font>
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*Glycerol stocks for the cultures, that were in their exponential growth phase (750 ul of bacteria and 250 ul of 80% glycerol).
*Glycerol stocks for the cultures, that were in their exponential growth phase (750 ul of bacteria and 250 ul of 80% glycerol).
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|[[Image:pv_manuel_pick.jpg|thumb|300px|left|Manuel picking colonies from plates.]]
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* We re-filled with 5 ml of LB + Amp the remaining 250 ul of the grown bacterial cultures containing:
* We re-filled with 5 ml of LB + Amp the remaining 250 ul of the grown bacterial cultures containing:
-
**J23100, J23101, J23118 (strong constitutive promoters with RFP protein generator downstream) to test fluorescence detection of the microplate reader;
+
**J23100, J23101, J23118 (strong constitutive promoters with RFP protein generator downstream) to test fluorescence detection of the microplate reader
**E0240 (negative control because bacteria bearing this BioBrick don't express fluorescent proteins)
**E0240 (negative control because bacteria bearing this BioBrick don't express fluorescent proteins)
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*We incubated the 5 ml cultures overnight at 37°C, 220 rpm.
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</td>
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<td>
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<font class='didascalia'>
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[[Image:pv_manuel_pick.jpg|thumb|200px|left|Manuel picking colonies from plates.]]
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</font>
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</td>
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</tr>
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</table>
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<div align="right">
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[[#top|Top]]
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== <html><font class="dayw_style">May, 27th</font></html> ==
== <html><font class="dayw_style">May, 27th</font></html> ==
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|I732017
|I732017
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*We diluted 1:10 the 5 ml overnight cultures of J23100, J23101, J23118, and E0240 in 5 ml of LB + Amp.
*We diluted 1:10 the 5 ml overnight cultures of J23100, J23101, J23118, and E0240 in 5 ml of LB + Amp.
*We incubated the diluted cultures at 37°C, 220 rpm for 4 and 1/2 hours.
*We incubated the diluted cultures at 37°C, 220 rpm for 4 and 1/2 hours.
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''Experiment with Tecan F200''
 
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* <html><u>Description</u></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>
 
<table width="100%">
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{|
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<font class='didascalia'>
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|[[Image:pv_susy_setup_at_tecan.jpg|thumb|300px|left|Susy setting up the first experiment with Tecan F200.]]
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[[Image:pv_susy_setup_at_tecan.jpg|thumb|300px|left|Susy setting up the first experiment with Tecan F200.]]
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</font>
</td>
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{|
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<font class='didascalia'>
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|[[Image:pv_first_microplate.jpg|thumb|300px|left|96-wells microplate at the end of the experiment: the grown cultures were turbid and RFP-expressing bacterial cultures were red-coloured.]]
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[[Image:pv_first_microplate.jpg|thumb|300px|left|96-wells microplate at the end of the experiment: the grown cultures were turbid and RFP-expressing bacterial cultures were red-coloured.]]
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== <html><font class="dayw_style">May, 28th</font></html> ==
== <html><font class="dayw_style">May, 28th</font></html> ==
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*All the five overnight plates showed colonies!
 +
 +
*We picked a colony for each plate to infect 1 ml of LB + Amp. We incubated the inocula at 37°C, 220 rpm for 5 and 1/2 hours.
 +
 +
*Glycerol stocks for the grown cultures.
 +
 +
*TBE 5X preparation.
 +
 +
*Colony PCR to check VF2 and VR primers quality. The primers had been ordered the previous year for iGEM 2008. We decided to perform this check on six colonies of E0240 plate.
 +
 +
*Electrophoresis for PCR reactions.
 +
 +
*Gel results (picture not available): no band could be seen on the gel (only the ladder)...Strange result, we will repeat the experiment tomorrow.
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<div align="right">
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[[#top|Top]]
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</div>
== <html><font class="dayw_style">May, 29th</font></html> ==
== <html><font class="dayw_style">May, 29th</font></html> ==
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<table width="100%">
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<tr valign="TOP">
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<td>
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*We repeated colony PCR for E0240 plate.
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 +
*Gel run for PCR vials.
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 +
*Gel results: primers worked correctly! all the bands have the expected size of 1114 bp. We will use the 2008 primer stocks.
 +
 +
*LB + Amp plates preparation.
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</td>
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<td>
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<font class='didascalia'>
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[[Image:pv_primer_check_PCR.jpg|thumb|300px|left|Colony PCR: E0240 insert was amplified correctly.]]
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</font>
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</td>
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</tr>
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</table>
<div align="right">
<div align="right">
[[#top|Top]]
[[#top|Top]]
</div>
</div>
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</div>
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<html>
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<hr/>
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<table width="100%">
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<tr>
 +
<td align="left" width="50%">
 +
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4May#week_start">
 +
<img src="https://2009.igem.org/Special:FilePath?file=Left_arrow.png"/>
 +
</a>
 +
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week4May#week_start">Previous Week</a>
 +
</td>
 +
<td align="right" width="50%">
 +
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Jun#week_start">Next Week</a>
 +
<a href="https://2009.igem.org/Team:UNIPV-Pavia/Notebook/Week1Jun#week_start">
 +
<img src="https://2009.igem.org/Special:FilePath?file=Right_arrow.png"/>
 +
</a>
 +
</td>
 +
</tr>
 +
</table>
 +
</html>

Latest revision as of 10:17, 21 October 2009

EthanolPVanimation.gif

December 2008
M T W T F S S
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8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30 31
March 2009
M T W T F S S
1
2 3 4 5 6 7 8
9 10 11 12 13 14 15
16 17 18 19 20 21 22
23 24 25 26 27 28 29
30 31
April 2009
M T W T F S S
1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30
May 2009
M T W T F S S
1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31
June 2009
M T W T F S S
1 2 3 4 5 6 7
8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30
July 2009
M T W T F S S
1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30 31
August 2009
M T W T F S S
1 2
3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31
September 2009
M T W T F S S
1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30
October 2009
M T W T F S S
1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30 31
November 2009
M T W T F S S
1
2 3 4 5 6 7 8
9 10 11 12 13 14 15
16 17 18 19 20 21 22
23 24 25 26 27 28 29
30

Week from May 25th, to May 31st, 2009

Previous Week Next Week

May, 25th

  • iGEM starts now!!!
  • We planned to dedicate the first two weeks to prepare a glycerol stock for every BioBrick of interest for our project. The following weeks will be dedicated to the BioBrick assembly.


  • DNA resuspension from iGEM 2009 plates. We resuspended all the high quality constitutive promoter family members from UCBerkeley collection and GFP protein generator:
J23100 J23101 J23102
J23103 J23104 J23105
J23106 J23110 J23112
J23113 J23114 J23116
J23117 J23118 E0240
  • Transformation of resuspended DNA (2 ul) in TOP10 E. coli, plated transformed bacteria and incubated the plates overnight at 37°C.

Top

May, 26th

  • All the 15 incubated plates showed colonies!

Moreover, many UCBerkeley promoter family members showed red fluorescent colonies, according to the ranking of promoters' estimated strength (see http://partsregistry.org/Promoters/Catalog/Anderson).

High quality Berkeley promoters and E0240 plates: high yield for all of them!

J23102 plate: the promoter is very strong and RFP expression could be seen in the red-coloured colonies. The same result was seen in the other strong Berkeley promoters.
  • We picked one colony from each plate and infected 1 ml of LB + Amp. We incubated the inocula at 37°C, 220 rpm for 5 and 1/2 hours.
  • After 5 hours and 1/2 all the cultures were turbid.
  • Glycerol stocks for the cultures, that were in their exponential growth phase (750 ul of bacteria and 250 ul of 80% glycerol).


Preparation of the first experiment with Tecan F200

  • We re-filled with 5 ml of LB + Amp the remaining 250 ul of the grown bacterial cultures containing:
    • J23100, J23101, J23118 (strong constitutive promoters with RFP protein generator downstream) to test fluorescence detection of the microplate reader
    • E0240 (negative control because bacteria bearing this BioBrick don't express fluorescent proteins)
  • We incubated the 5 ml cultures overnight at 37°C, 220 rpm.

Manuel picking colonies from plates.

Top

May, 27th

  • We wanted to resuspend Q04400 and Q04121 in order to build up respectively an aTc and an IPTG/lactose sensor, but we realized that MIT QC classified their sequences as "inconsistent"...
  • DNA resuspension from iGEM 2009 plates. We resuspended three lysis-involved parts, a double transcriptional terminator and lacZ transcriptional unit in order to build up a beta-galactosidase protein generator (last year, a beta-galactosidase protein generator was available in 2008 Distributon - I732950 - but its sequence was almost completely wrong):
K131009 (colE2 operon without promoter) K117000 (colE7 lysis gene) K124017 (bacteriophage lysis cassette coding sequence)
B0015 I732017
  • Only B0015 and I732017 QC sequence analysis was available...we hoped that the three lysis parts' sequences were correct!


  • Transformation of resuspended DNA (2 ul) in TOP10 E. coli, plated transformed bacteria and incubated the plates overnight at 37°C.


  • We planned how to substitute Q04400 and Q04121. We searched for smaller BioBricks to re-build them with a minimum number of assemblies:
    • Q04400 = P0440 (available in iGEM 2009 plates) + R0040 (available in iGEM 2009 plates)
    • Q04121 = P0412 (not available in iGEM 2009 plates) + R0011 (available in iGEM 2009 plates)


Preparation of the first experiment with Tecan F200

  • We diluted 1:10 the 5 ml overnight cultures of J23100, J23101, J23118, and E0240 in 5 ml of LB + Amp.
  • We incubated the diluted cultures at 37°C, 220 rpm for 4 and 1/2 hours.

Susy setting up the first experiment with Tecan F200.

96-wells microplate at the end of the experiment: the grown cultures were turbid and RFP-expressing bacterial cultures were red-coloured.

Top

May, 28th

  • All the five overnight plates showed colonies!
  • We picked a colony for each plate to infect 1 ml of LB + Amp. We incubated the inocula at 37°C, 220 rpm for 5 and 1/2 hours.
  • Glycerol stocks for the grown cultures.
  • TBE 5X preparation.
  • Colony PCR to check VF2 and VR primers quality. The primers had been ordered the previous year for iGEM 2008. We decided to perform this check on six colonies of E0240 plate.
  • Electrophoresis for PCR reactions.
  • Gel results (picture not available): no band could be seen on the gel (only the ladder)...Strange result, we will repeat the experiment tomorrow.

Top

May, 29th

  • We repeated colony PCR for E0240 plate.
  • Gel run for PCR vials.
  • Gel results: primers worked correctly! all the bands have the expected size of 1114 bp. We will use the 2008 primer stocks.
  • LB + Amp plates preparation.

Colony PCR: E0240 insert was amplified correctly.

Top

Previous Week Next Week

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