Team:UNIPV-Pavia/Notebook/Week3Aug

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<html><a name="week_start"></a></html>
= Week from August 17th, to August 23rd, 2009 =
= Week from August 17th, to August 23rd, 2009 =
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== <html><font class="dayw_style">August, 17th</font></html> ==
== <html><font class="dayw_style">August, 17th</font></html> ==
 +
 +
*This week we planned to perform a gel run for all the constructs of the ethanol producing operon in order to check for contaminant bands. Then, we planned to ligate a promoter upstream of the ethanol producing operon and to build up measurement systems for A11 (lactose sensor) and A12 (aTc sensor).
 +
 +
 +
 +
*We inoculated 8 ul of:
 +
**B1-13
 +
**B2-5
 +
**B3-5
 +
**B4-2
 +
**B5-3
 +
**B6-3
 +
**E0240
 +
**A11-2
 +
*glycerol stocks in 5 ml of LB + suitable antibiotic.
 +
 +
*We incubated these cultures overnight (37°C, 220 rpm).
 +
 +
 +
 +
 +
 +
''Preparation of experiment with Tecan F200''
 +
 +
*We dissolved 22 mg of lactose in 5 ml of LB + Kan in order to have 4.5% lactose.
 +
 +
*We prepared lactose assay kit for testing.
 +
 +
 +
 +
 +
 +
''Experiment with Tecan F200''
 +
 +
* <html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/Lactose Standard Cruve 17-08-09.pdf" target="_blank">Download Protocol</a></html>
<div align="right">
<div align="right">
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== <html><font class="dayw_style">August, 18th</font></html> ==
== <html><font class="dayw_style">August, 18th</font></html> ==
 +
*Glycerol stock for E0240, A11-2, B1, B2, B3 and B4 in order to have a backup.
 +
 +
*Miniprep for:
 +
**B1-13
 +
**B2-5
 +
**B3-5
 +
**B4-2
 +
**B5-3
 +
**B6-3
 +
**E0240
 +
**A11-2
 +
 +
*Digestion with EcoRI for:
 +
**B1-13
 +
**B2-5
 +
**B3-5
 +
**B4-2
 +
**B5-3
 +
**B6-3
 +
 +
*Digestion with EcoRI-PstI for:
 +
**B1-13
 +
**B2-5
 +
**B3-5
 +
**B4-2
 +
**B5-3
 +
**B6-3
 +
 +
*Digestion with PstI for:
 +
**B1-13
 +
**B2-5
 +
**B3-5
 +
**B4-2
 +
**B5-3
 +
**B6-3
 +
 +
*Electrophoresis for the digested plasmids.
 +
 +
<font class='didascalia'>
 +
{|align="center"
 +
|[[Image:pv_B123456summary.jpg|thumb|500px|left|Digestion check on fermentation parts (1st gel).]]
 +
|-
 +
|[[Image:pv_B123456summary2.jpg|thumb|500px|left|Digestion check on fermentation parts (2nd gel).]]
 +
|}
 +
</font>
 +
 +
*Gel results:
 +
**B1 - ok
 +
**B2 - ok
 +
**B3 - two extra-bands (consistent with previous gels)
 +
**B4 - ok
 +
**B5 - two extra bands (consistent with previous gels)
 +
**B6 - two extra bands (consistent with previous gels)
 +
 +
*We decided to try to ligate a promoter upstream of B5 and B6 anyway.
 +
 +
 +
*We inoculated 8 ul of:
 +
**A4(X2)
 +
**A12
 +
*glycerol stocks.
 +
 +
*We incubated these inocula at 37°C, 220 rpm overnight.
<div align="right">
<div align="right">
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*Precipitation with sodium acetate for:
*Precipitation with sodium acetate for:
**A11-2(S-P)
**A11-2(S-P)
 +
**B5(E-X)
**B6(E-X)
**B6(E-X)
-
**B7(E-X)
 
**A4(E-S)(X2)
**A4(E-S)(X2)
**A12(S-P)
**A12(S-P)
-
*Gel run/cur/purification for E0240.
+
*Gel run/cut/purification for E0240.
*Ligations:
*Ligations:
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**A14 = A11(S-P) + E0240(X-P) in pSB1A2
**A14 = A11(S-P) + E0240(X-P) in pSB1A2
**A16 = A12(S-P) + E0240(X-P) in pSB1A2
**A16 = A12(S-P) + E0240(X-P) in pSB1A2
 +
 +
 +
''Experiment with Tecan F200''
 +
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/T9002 induction test TEST 19-08-09.pdf" target="_blank">Download Protocol</a></html>
<div align="right">
<div align="right">
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*We resuspended pSB3K3 from 2009 Registry Distribution. The plasmid with ccdB was not consistent, so we resuspended a consistent brick (of ~700bp length, suggested in the Registry "Help" page: Kit Plate 2, well 15L, I714891 brick) contained in pSB3K3.
*We resuspended pSB3K3 from 2009 Registry Distribution. The plasmid with ccdB was not consistent, so we resuspended a consistent brick (of ~700bp length, suggested in the Registry "Help" page: Kit Plate 2, well 15L, I714891 brick) contained in pSB3K3.
-
*We transformed the overnight ligations and pSB3K3 in TOP10 and plated transformed bacteria on LB agar plates + Amp (A14 and A16) or + Kan (pSB3K3, B7 and B8). We incubated the plates at 37°C overnight.
+
*We transformed the overnight ligations and I714891 in TOP10 and plated transformed bacteria on LB agar plates + Amp (A14 and A16) or + Kan (pSB3K3, B7 and B8). We incubated the plates at 37°C overnight.
 +
 
 +
 
 +
 
 +
*We sent these samples (stored at -20°C) to BMR Genomics for sequencing:
 +
**A15-1
 +
**A15-3
 +
**A11-2
 +
**A11-3
 +
**B5-3
 +
**B6-3
 +
**B3-5 (again, because we wanted to check for contaminants in our native stock)
<div align="right">
<div align="right">
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*Electrophoresis for PCR results.
*Electrophoresis for PCR results.
-
<font class='didascalia'>
+
*Gel results (picture not taken, sorry...):
-
{|align="center"
+
-
|[[Image:pv_.jpg|thumb|500px|left|Insert here]]
+
-
|}
+
-
</font>
+
-
 
+
-
*Gel results:
+
**A14 - 3rd colony seems the most pure and it had the expected length for ligated plasmid.
**A14 - 3rd colony seems the most pure and it had the expected length for ligated plasmid.
**A16 - reaction worked only on A16-1, but it was negative.
**A16 - reaction worked only on A16-1, but it was negative.
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-
*We picked 2 colonies from B7, 5 colonies from B8 and 1 colony from pSB3K3 plates. We inoculated them in 1 ml of LB + Kan and incubated them at 37°C, 220 rpm for 5 hours and 1/2. Then we prepared glycerol stocks and re-filled the remaining 250 ul with 4 ml of LB + Kan.
+
*We picked 2 colonies from B7, 5 colonies from B8 and 1 colony from I714891 plates. We inoculated them in 1 ml of LB + Kan and incubated them at 37°C, 220 rpm for 5 hours and 1/2. Then we prepared glycerol stocks and re-filled the remaining 250 ul with 4 ml of LB + Kan.
*NOTE: we decided not to perform PCR on B7 and B8 plates because of the large size of positive inserts.
*NOTE: we decided not to perform PCR on B7 and B8 plates because of the large size of positive inserts.
*We incubated the re-filled cultures at 37°C, 220 rpm overnight.
*We incubated the re-filled cultures at 37°C, 220 rpm overnight.
 +
 +
 +
''Experiment with Tecan F200''
 +
*<html><a href="http://aimed11.unipv.it/iGEM2009/TestTECANInfiniteF200/HSL induction test LB M9 TEST 21-08-09 .pdf" target="_blank">Download Protocol<a></html>
 +
 +
<div align="right">
 +
[[#top|Top]]
 +
</div>
 +
 +
== <html><font class="dayw_style">August, 22nd</font></html> ==
 +
 +
*Pellet preparation for the 10 overnight cultures, ready to be miniprepped next week! Pellets were stored at -20°C.
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Latest revision as of 15:15, 20 October 2009

EthanolPVanimation.gif

December 2008
M T W T F S S
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29 30 31
March 2009
M T W T F S S
1
2 3 4 5 6 7 8
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16 17 18 19 20 21 22
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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
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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 August 17th, to August 23rd, 2009

Previous Week Next Week

August, 17th

  • This week we planned to perform a gel run for all the constructs of the ethanol producing operon in order to check for contaminant bands. Then, we planned to ligate a promoter upstream of the ethanol producing operon and to build up measurement systems for A11 (lactose sensor) and A12 (aTc sensor).


  • We inoculated 8 ul of:
    • B1-13
    • B2-5
    • B3-5
    • B4-2
    • B5-3
    • B6-3
    • E0240
    • A11-2
  • glycerol stocks in 5 ml of LB + suitable antibiotic.
  • We incubated these cultures overnight (37°C, 220 rpm).



Preparation of experiment with Tecan F200

  • We dissolved 22 mg of lactose in 5 ml of LB + Kan in order to have 4.5% lactose.
  • We prepared lactose assay kit for testing.



Experiment with Tecan F200

Top

August, 18th

  • Glycerol stock for E0240, A11-2, B1, B2, B3 and B4 in order to have a backup.
  • Miniprep for:
    • B1-13
    • B2-5
    • B3-5
    • B4-2
    • B5-3
    • B6-3
    • E0240
    • A11-2
  • Digestion with EcoRI for:
    • B1-13
    • B2-5
    • B3-5
    • B4-2
    • B5-3
    • B6-3
  • Digestion with EcoRI-PstI for:
    • B1-13
    • B2-5
    • B3-5
    • B4-2
    • B5-3
    • B6-3
  • Digestion with PstI for:
    • B1-13
    • B2-5
    • B3-5
    • B4-2
    • B5-3
    • B6-3
  • Electrophoresis for the digested plasmids.

Digestion check on fermentation parts (1st gel).
Digestion check on fermentation parts (2nd gel).

  • Gel results:
    • B1 - ok
    • B2 - ok
    • B3 - two extra-bands (consistent with previous gels)
    • B4 - ok
    • B5 - two extra bands (consistent with previous gels)
    • B6 - two extra bands (consistent with previous gels)
  • We decided to try to ligate a promoter upstream of B5 and B6 anyway.


  • We inoculated 8 ul of:
    • A4(X2)
    • A12
  • glycerol stocks.
  • We incubated these inocula at 37°C, 220 rpm overnight.

Top

August, 19th

  • Digestion for:
    • E0240(X-P)
    • A11-2(S-P)
    • B5(E-X)
    • B6(E-X)
  • Miniprep for:
    • A4(X2)
    • A12
  • Digestion for:
    • A4(E-S)(X2)
    • A12(S-P)
  • Precipitation with sodium acetate for:
    • A11-2(S-P)
    • B5(E-X)
    • B6(E-X)
    • A4(E-S)(X2)
    • A12(S-P)
  • Gel run/cut/purification for E0240.
  • Ligations:
    • B7 = A4(E-S) + B5(E-X) in pSB1AK3
    • B8 = A4(E-S) + B6(E-X) in pSB1AK3
    • A14 = A11(S-P) + E0240(X-P) in pSB1A2
    • A16 = A12(S-P) + E0240(X-P) in pSB1A2


Experiment with Tecan F200

Top

August, 20th

  • We resuspended pSB3K3 from 2009 Registry Distribution. The plasmid with ccdB was not consistent, so we resuspended a consistent brick (of ~700bp length, suggested in the Registry "Help" page: Kit Plate 2, well 15L, I714891 brick) contained in pSB3K3.
  • We transformed the overnight ligations and I714891 in TOP10 and plated transformed bacteria on LB agar plates + Amp (A14 and A16) or + Kan (pSB3K3, B7 and B8). We incubated the plates at 37°C overnight.


  • We sent these samples (stored at -20°C) to BMR Genomics for sequencing:
    • A15-1
    • A15-3
    • A11-2
    • A11-3
    • B5-3
    • B6-3
    • B3-5 (again, because we wanted to check for contaminants in our native stock)

Top

August, 21st

  • Colony PCR for A14 (5 colonies) and A16 (5 colonies) plates. Colonies were inoculated in 1 ml of LB + Amp and let grow waiting for the end of the reaction.
  • Electrophoresis for PCR results.
  • Gel results (picture not taken, sorry...):
    • A14 - 3rd colony seems the most pure and it had the expected length for ligated plasmid.
    • A16 - reaction worked only on A16-1, but it was negative.
  • We decided to keep A14-3 (positive at PCR) and A16-4 (randomly chosen) for digestion screening: we prepared a glycerol stock for them and re-filled the remaining 250 ul of bacteria with 4 ml of LB + Amp.
  • We incubated these cultures at 37°C, 220 rpm overnight.



  • We picked 2 colonies from B7, 5 colonies from B8 and 1 colony from I714891 plates. We inoculated them in 1 ml of LB + Kan and incubated them at 37°C, 220 rpm for 5 hours and 1/2. Then we prepared glycerol stocks and re-filled the remaining 250 ul with 4 ml of LB + Kan.
  • NOTE: we decided not to perform PCR on B7 and B8 plates because of the large size of positive inserts.
  • We incubated the re-filled cultures at 37°C, 220 rpm overnight.


Experiment with Tecan F200

Top

August, 22nd

  • Pellet preparation for the 10 overnight cultures, ready to be miniprepped next week! Pellets were stored at -20°C.

Top

Previous Week Next Week
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