Melbourne/Lab Notebook Weeks 9-12


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Week 9

19 Aug 2007

  • Picked 3-4 transformants from "ligation" plates (17/8) and Grew up liquid cultures.
  • Transformed the two ligation reactions in the pink rack in the cold room and plated on Gen + Amp plate.

20 Aug 2007

Desired products:

    1. 450bp (95bp different from CI-2 used as control)
    2. 450bp
    3. 450bp
    4. 1kb
    5. 1kb
  • These were run on a gel.

-Vector: OmpR (AmpR) miniprepped P1 15P from 10/7 digested with S/P.
-Insert: c1 inverter (1kb fragment) P2 13K A with X/P.

-Vector: LacZ (KanR) P2 9E from 6/8 with E/X.
-Insert: RBS (GenR) P4 8J from 6/7 with E/S.

Digested for 2h and heat inactivated for 10min at 80degC.

21 Aug 2007

  • Picked 4 colonies from fresh ligation/transformation to Growing up.

- These transformations were made as follows:

    • Ligated
      1. 5ul of Purified insert from 17/8 (CI-2 E/S)
      2. 5ul of Redigested P1 5G with E/X and heat inactivated at 60degC for 10min.

22 Aug 2007

  • Miniprepped L1-L4 (liquid cultures from 21/8).
  • Digested these for ligations.
    1. L1 X/P
    2. L2 X/P
    3. L3 X/P
    4. L4 X/P
    5. CI-2 X/P (control for 350bp)
    6. P1 15P S/P

All L1-L4 had the 450bp desired band.

  • Gel purified 450bp band from L3 and a 950bp band from the P2 13K digest.

OmpR-L3 (RBS-LacZa-double term)
-Vector= P1 15P S/P (above)
-Insert= L3 X/P (above)

OmpR-c1 inverter (P2 13K)
-Vector= P1 15P S/P (above)
-Insert= P1 15P S/P (above)

Ligations were kept overnight at 4degC.

23 Aug 2007

  • Transformed ligations from 22/8 into competent NM522 cells.

24 Aug 2007

  • Liquid cultures were made of transformations from 23/8. 6 colonies were picked from each ligation.

25 Aug 2007

  • Ran on a Gel 16 samples that were miniprepped.
  • Gel purified 1000bp fragment from ompR-c1 inverter ligation digest and a 600bp fragment from ompR-L3 ligation digest.

Week 10

26 Aug 2007

(all from 25/8) Left overnight at 4degC.

27 Aug 2007

Plated on Amp plates.

28 Aug 2007

  • Picked six colonies from each plate (27/8) and cultured at 37degC for 13 hours.

29 Aug 2007

-The M30109 ligations yielded random fragments: something wrong with the part? - P1 15P-L3 and P1 15P-c1 inverter ligations had bands of the desired size.

30 Aug 2007

  • Set up minipreps from 29/8 for sequencing.

OmpR-L3 and OmpR-c1 inverter were the desired sequences.

Parts created:

OmpR-RBS-LacZa-double terminator

OmpR-c1 inverter

31 Aug 2007

1 Sept 2007

Week 11

2 Sept 2007

    1. EcoRV
    2. Xho1
    3. EcoRV/Xho1
    4. E/P
    5. E/S
    6. E/X
    7. X/P
    8. X/S
    9. S/P
    10. E
    11. P
    12. S
    13. EcoR1/EcoRV/Xho1
    14. Apa1
    15. BamH1
    16. EcoR1/BamH1

The results showed that although a fragment of cph8 may be present in the part (1200bp fragment present only when double digested by EcoRV/Xho1), it may not be in the form as designated in the iGEM wiki.

The original miniprep was sent off for sequencing using vf2. The results showed that the part is a form of RBS-cph8, although the sequencing got cut off in the middle of the cp8 sequence. Therefore, the entire part designated is not present. We consequently decided to create the M30109 part ourselves.

3 Sept 2007

4 Sept 2007

5 Sept 2007

  • Recieved ComP and ComA sequences from GeneArt.

6 Sept 2007

  • Transformed the following on Amp plates except where labeled
    1. ComA
    2. ComP (Kan plate)
    3. 6kb band from undigested M30109
    4. 10kb band from undigested M30109
    5. RBS-LacZa-double terminator from 22/8
    6. OmpR-RBS-LacZa-double terminator from 29/8
    7. OmpR-RBS-LacZa-double terminator from 29/8 into CP919 EnvZ- strains.

7 Sept 2007

  • Liquid cultured a colony from each of the 6/9 transformations. No growth was observed for the M30109 undigested plates and were discarded.

8 Sept 2007

-All had desired sizes.

Week 12

9 Sept 2007

ComP-backbone vector
-Vector: P3 20I Death Plasmid AmpR X/P fragment from 8/9.
-Insert: ComP X/P fragment from 8/9.

ComA-backbone vector
-Vector: P3 20I Death Plasmid AmpR X/P fragment from 8/9.
-Insert: ComA X/P fragment from 8/9.

10 Sept 2007

11 Sept 2007

12 Sept 2007

and minipreprs from previous dates:

  • Efficiency of fast ligations were simultaneously tested for. Using 2X ligase buffer, 2hour and 5min ligations were tested using ComA parts from 8/9. These did not have ligation efficiencies as high as the overnight ligations but were sufficient. The 5 min ligation seemed to have more colonies than the 2 hour ligation.

13 Sept 2007

  • P4 8J and P1 5G were overgrown so were streaked onto new plates.
  • Colony PCR using The Endy Procol [1] of 2 colonies from ComA-cell death (12/9) and OmpR-L3 (27/8),
    1. 5ul 10 X PCR buffer (green)
    2. 5ul MgCl2 25mM
    3. 0.5ul vf2
    4. 0.5ul vR
    5. 1.0ul dNTPs 25mM
    6. 0.5ul GoTaq
    7. 37.5ul milliQ water

on a cycle of:

    1. 94C for 5min
    2. 94C for 30 seconds
    3. 55C for 30 seconds
    4. 72C for 1:15 minutes
    5. repeat 2-4 for 30 times
    6. 72C for 7min.

14 Sept 2007

15 Sept 2007