Team:UNIPV-Pavia/Notebook/Week1Aug

From 2009.igem.org

Revision as of 11:47, 8 August 2009 by Lor18 (Talk | contribs)

EthanolPVanimation.gif

December 2008
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
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 August 3rd, to August 9th, 2009

Previous Week Next Week

August, 3rd

  • This week we planned to continue the assembly of the synthetic ethanologenic operon.


  • We received DH5alpha, XL1-Blue and DB3.1 competent cells from Bologna iGEM Team. Thank you!!


  • We performed 2 screenings for the 7 samples of B3 and for the 7 samples of B4:
  • SCREENING 1 - Ojective: check the presence of non-ligated plasmids. We planned not to digest with standard enzymes because the resulting fragments would result either too small or too big. Insert excision would generate a possible 129pb fragment, while vector linearization would generate possible 3Kb or 5Kb fragments. We used ApE to decide the best enzymes to cut. Digestion with XhoI-PstI (6 ul of DNA, final volume 20ul).
  • Gel results:
    • Only B3-1 and B3-5 showed the bands with expected length for the correct plasmid.
    • All B4 samples showed the bands with expected lengths!
  • SCREENING 2 - Ojective: check the presence of the right insert length for B3 samples, because there was the possibility that B1 vector (pSB1A2) ligated in B0015 vector (pSB1AK3) as an insert. If so, no XbaI site is present in the final plasmid, while correct ligations should show a ~1900bp fragment as an insert. Digestion with XbaI-PstI (2 ul of DNA, final volume 20ul). We decided to perform the reaction for all B3 samples, even if only two were good.
  • Gel results: B3-1 and B3-5 showed the expected bands for pSB1AK3 (~3200 bp) and B1-B0015 ligated insert (~1900bp).
  • We sent the following purified DNA samples to BMR Genomics for sequencing:
    • B3-1
    • B3-5
    • B4-2
    • B4-4


  • LB agar plates + Kan preparation.



Preparation of experiment with Tecan F200

  • We inoculated 10 ul of A1, A2, A7, J23100, J23101 and J23118 glycerol stocks in 5 ml of LB + Amp, while we used a single colony from B0030 native plate to infect 5 ml of LB + Amp.
  • We incubated these inocula overnight (37°C, 220 rpm).

Top

August, 4th

Top

August, 5th

Top

August, 6th

  • We received Ethanol Assay Kit and Lactose Assay Kit from BioVision.

Top

August, 7th

  • Miniprep for:
B1-13 (X2) B2-5 (X2) B3-5 (X2)
B4-2 (X2) R0011 F2620MIT1
BOL1 K112808
  • We stored purified DNA at -20°C and next week we will perform digestion for these 12 samples, in order to: i) finish the assembly of the ethanologenic operon, ii) re-assemble A11, which sequence analysis showed a deletion and iii) build up an inducible lysis device.

Top




Previous Week Next Week