From 2007.igem.org

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• Weeks 1-4
• Weeks 5-8
• Weeks 9-12
• Weeks 13-16
• Weeks 17-18

Contents 1 Week 1 2 Week 2 3 Week 3 4 Week 4 5 Week 5 6 Week 6 7 Week 7 8 Week 8 9 Week 9 10 Week 10 11 Week 11 12 Week 12 13 Week 13 14 Week 14 15 Week 15 16 Week 16 17 Week 17 18 Week 18

Week 1

• 25 June 2007: Prepared LB agar plates Amp & Kana.
• 25 June 2007: Resuspended the following from Registry plates & Transformed (shorter protocol) into competent DH5alpha cells.
  1. P2 21A - (BBa_I15008, ho1, Kan) -> No colonies on plates
  2. P2 21C - (BBa_I15009, PcyA, Kan) ->No colonies on plates
  3. P1 11H - (BBa_R0084, OmpR positive promoter, Amp)->Small number of colonies.

• 26 June 2007: Resuspended the following from Registry plates & Transformed into Joe's competent DH5alpha cells.
  1. P1 3O (BBa_B0034, RBS, Amp)
  2. P1 5G (BBa_C0051, c1 protein, Amp)
  3. P2 3P (BBa_B0010, Terminator, Amp)

• 26 June 2007: Streaked the following cells:
  1. pJS010 (from solid agar, Amp)
  2. Fusion protein (from glycerol stock, Amp?)
  3. BBa_I15010 (from solid agar, Kan)

• 27 June 2007: Transformed into Joe's competent DH5alpha cells.
  1. P2 21A (Kan)
  2. P2 21C (Kan)

Liquid culture

1. Prepared 30mL of Amp LB (100ug/mL). Prepared 5mL Kan LB (50mg/mL).
2. Aliquoted 5mL Amp LB into 6 50mL falcon tubes
3. To the Amp LB aliquots single transformed colonies from plates of the following were introduced:
   • pJS010
   • Fusion
   • P1 3O
   • P1 5G
   • P2 3P
   • P1 11H
4. To the Kan LB a single colony from the transformation plate of BBa_I15010 was introduced.
5. Cells incubated at 37degrees with shaking overnight.

28 June 2007

Miniprep

1. Minipreped the cultures grown overnight after putting aside 1mL(sterile) for glycerol stocks:
   • pJS010
   • Fusion
   • P1 3O
   • P1 5G
   • P2 3P
   • P1 11H
   • I15010
2. Stored in -20 freezer

Liquid culture

1. Cultured the following cells from transformed plates:
   • P1 11H (BBa_R0084)
   • BBa_I15010
   • P1 3O (BBa_B0034)
   • P1 5G (BBa_C0051)
   • P1 21A (BBa_I15008)
   • P1 21C (BBa_I15009)

29 June 2007
Miniprep

1. Minipreped the cultures grown overnight after putting aside 1mL(sterile) for glycerol stocks:
   • P1 11H (BBa_R0084)
   • BBa_I15010
   • P1 3O (BBa_B0034)
   • P1 5G (BBa_C0051)
   • P1 21A (BBa_I15008)
   • P1 21C (BBa_I15009)

1. Stored in -20 freezer

Week 2

• 2 July 2007: Resuspended the following from Registry plates & Transformed into Joe's competent DH5alpha cells.
  1. Q04510
  2. E0241
  3. E0040
  4. B0014
  5. J61035

• 3 July 2007: Re Transformed into Joe's competent DH5alpha cells. Each Plate had additional 40uL of 50mg/ml Ampicillan spread on surface.
  1. P4 8J -> Three colonies -> grew in liquid culture 4 july
  2. P1 5H -> Multiple colonies at edge -> did not grow in overnight liquid culture 4 july 'poor amp spreading'
  3. P2 15L -> Three colonies -> grew in liquid culture 4 july

• 4 July 2007:

Ampicillin Plates

• Made up 25 Ampicillin plates using 400mL LB agar prepared on the 25th of June.
  • Used 100mg/ml Ampicillin stock from Gooley Lab - In bag in left freezer near our lab bench.

Transformation
Transformed the following and grew on new ampicillin plates

• P1 5H
• P4 8J
• P2 15L
  • Also Plated an untransformed control (subjected to transformation protocol in absense of DNA) and a blank plate control (no cells)

Liquid Culture

• Cultured 2 colonies from each of the following transformed plates and labelled as follows
  • P2 3P 1
  • P2 3P 2
  • I15010 1
  • I15010 2
  • pJS010 1
  • pJS010 2
  • Fusion 1
  • Fusion 2
  • P4 8J 1
  • P4 8J 2
  • P2 15L 1
  • P2 15L 2
  • P2 13K 1
  • P2 13K 2
  • P1 1G 1
  • P1 1G 2
  • P1 11H 1
  • P1 11H 2
  • P1 5H 1
  • P1 5H 2
  • suspected contaminating colony from P4 8J

5 July 2007

Miniprep

• 1mL culture from the 4th of Julyput aside for glycerol stocks under sterile conditions. Labelled with todays date 5/7
• miniprepped the following overnight cultures set up on the 4th of July. Final elution was performed with TE buffer prepared on 25/06/07 rather than nuclease free water. Labelled with todays date 5/7.
  • P2 3P 1
  • P2 3P 2
  • I15010 1
  • I15010 2
  • pJS010 1
  • pJS010 2
  • Fusion 1
  • Fusion 2
  • P4 8J 1
  • P4 8J 2
  • P2 15L 1
  • P2 15L 2
  • P2 13K 1(eluted in 130uL due to accidental double application of 50uL elution)
  • P2 13K 2
  • P1 1G 1
  • P1 1G 2
• the following liquid cultures were not miniprepped due to failure (no growth)
  • P1 11H 1
  • P1 11H 2
  • P1 5H 1
  • P1 5H 2
  • suspected contaminating colony from P4 8J

Digest
Performed the following digests on DNA from the above miniprep

EcoR1/Pst1 with buffer 3

• I15010 1
• I15010 2
• P4 8J 1
• P4 8J 2
• P2 15L 1
• P2 15L 2
• P2 13K 1
• P2 13K 2

EcoR1/HaeII in buffer 2

• P2 3P 1
• P2 3P 2
• P1 1G 1
• P1 1G 2

XbaI/SpeI in buffer 2

• pJS010 1
• pJS010 2

Incubated for 2hours at 37 degrees, before addition of loading dye and storage at -20

Transformation

• Transformed P1 11H from resuspended DNA.

Liquid Culture

• P1 5H 1
• P1 5H 2
• P4 8J 1
• P4 8J 2
• P2 15L 1
• P2 15L 2

6 July 2007

Digest Gel

• Prepared 20 lane 100mL agarose gel with 0.5xTBE buffer.
• Loaded 20uL of digest samples in the following lane order
  1. 1kb+ ladder
  2. I15010 1
  3. I15010 2
  4. P2 13K 1
  5. P2 13K 2
  6. P2 15L 1
  7. P2 15L 2
  8. P4 8J 1
  9. P4 8J 2
  10. P1 1G 1
  11. P1 1G 2
  12. P2 3P 1
  13. P2 3P 2
  14. pJS010 1
  15. pJS010 2
• Ran for 1.5hours at 95V

Miniprep

• Put aside 1mL of liquid cultures set up on the 5th for glycerol stocks and labelled with todays date 6/7
• Miniprepped the remains of the cultures and labelled with todays date 6/7. Samples were eluted with TE buffer.
• P1 5H 1
• P1 5H 2
• P4 8J 1
• P4 8J 2
• P2 15L 1
• P2 15L 2

Digest

• Digested 5uL of each of the above miniprep DNA with EcoRI and PstI in buffer 3.
• Incubated for 2hours 25min at 37degrees
• Added 5uL 6x loading dye and stored at -20

Glycerol Stocks
The following glycerol stocks were made:

• Put aside from cultures 5/7 (labelled with this date)
  1. P2 15L 1
  2. P2 15L 2
  3. P4 8J 1
  4. P4 8J 2
  5. P1 1G 1
  6. P1 1G 2
• Put aside from cultures 6/7 (labelled with this date)
  1. P2 15L 1
  2. P2 15L 2
  3. P4 8J 1
  4. P4 8J 2
  5. P1 5H 1
  6. P1 5H 2

Stored at -80

Liquid Culture
Cultured the following in 5mL LB

• I15010 1 (Kan)
• I15010 2
• P1 11H 1 (Amp)
• P1 11H 2

Also placed Kan plate in the incubator to test antibiotic efficiency - no growth on 8/7

7 July 2007

• Made 10x TAE buffer

Digest Gel

• Prepared 8 lane 60mL agarose gel with 1xTAE buffer.
• Loaded 20uL of digest samples from 6/7 in the following lane order.
  1. P4 8J 1
  2. P4 8J 2
  3. P1 5H 1
  4. P1 5H 2
  5. P2 15L 1
  6. P2 15L 2

Glycerol Stocks
The following glycerol stocks were made and dated 7/7:

• I15010 1
• I15010 2
• P1 11H 1
• P1 11H 2

Stored at -80

Miniprep

• Miniprepped the remains of the cultures and labelled with todays date 7/7. Samples were eluted with TE buffer.

Digest

• Digested 5uL of each of the above miniprep DNA
  • EcoRI/PstI in buffer 3
    • I15010 1 (Kan)
    • I15010 2
    • P1 11H 1 (Amp)
    • P1 11H 2
• Incubated for 3hours at 37degrees
• Added 5uL 6x loading dye and stored at -20

8 July 2007

Transformation
Resuspended and transformed the following

• P1 15P(BBa_R0082, Omp R+, Amp)
• P1 17H(BBa_R0083, truncated BBa_R0082 Omp R+, Amp)
• P1 11A(BBa_E0430, EYFP(RBS+,LVA-,term) Amp)
• P1 16E(BBa_E0430; RBS,GFP,term; Amp)

The following was also retransformed due to colonies on previous plate appearing to be contaminants and failure of plasmid isolation from these colonies.

• P2 13K (BBa_Q04510, c1 inverter, Kan)

Week 3

9 July 2007

• Digested I15010(E/P),P1-11H(E/H),P2-15L 1.5hrs run
• liquid culture P1-11H,I15010,P1-15P,P1-16E,P1-17H,P2-13K

10 July 2007

• Miniprep
• Digest P1-16E,P1-11A,P2-13K,I15010,P1-15P,P1-11H,P1-17H 1.0hrs run
• liquid culture I15010, P2

11 July 2007

• Digest for ligation P1-15P(1)10/7,P1-11H 10/7,P1-17H(1)10/7,P1-16E(2)10/7,P1-11A(1)10/7
• loaded order X/P P1-11A,X/P P1-16E,S/P P1-15P,S/P P1-11H
  • Spe1(6uL)/Pst1(7.5uL)/AP(1.5uL), Buffer2 9uL, BSA 9uL, milliQ 27uL into 20uL aliquots with 10uL DNA.
  • Xbal1(3uL)/PstI(4uL),Buffer2 6uL,10XBSA 6uL,milliQ 21uL into 20uL aliquots with 10uL DNA
• Excise bands of interest and purify invitrogen
• liquid culture P1-11A,P1-15P 10ml
• Transform P2-21B,P2-23N,P3-20I into DB3.1 heat shock.
• Glycerol stocks P1-11A,P1-16E,P1-11H,P1-15P,P1-17H

12 July 2007

• Ran Gel P1-11A,P1-16E,P1-15P,P1-11H
• miniprepped P1-11A,P1-15P
• Digest
• Ligate control=(2uL ligase buffer,1uL ligase,5uL vector(P1-15P),12uL H20)
• Ligate (2uL ligase buffer,1uL ligase,5uL vector(P1-15P),10uL insert(P1-11A),2uL H20)
• Liquid culture transformants 11/7

13 July 2007

• miniprep cultures from transformants 11/7
• Digestion P1-15P and P1-11A from 12/7/07 37degC 3hours 15 minutes stopped 5uL of 6X loading dye.
• Excise bands 800bp from P1-11A, 2Kbp from P1-15P and purified.
• Glycerol stocks of P3-20I,P2-21B,P2-23N
• Transform DH5a with ligation product

14 July 2007

• Transform using 10uL of ligation reaction

Week 4

16 July 2007

• Colony PCR

18 July 2007

• Purchased XbaI,EcoRI,PstI
• Miniprepped cultures 1,3,6 from 16/7
• Digestion with E/P
• Ran Gel:std,ctrl(from PCR reaction 1 earlier),Digest1,3,6,(P2-15P)ages ago,(P1-11A)ages ago?

Week 5

23 July 2007

24 July 2007

25 July 2007

26 July 2007

27 July 2007

Week 6

30 July 2007

31 July 2007

1 Aug 2007

2 Aug 2007

3 Aug 2007

4 Aug 2007

Week 7

5 Aug 2007

6 Aug 2007

7 Aug 2007

8 Aug 2007

9 Aug 2007

10 Aug 2007

11 Aug 2007

Week 8

12 Aug 2007

13 Aug 2007

14 Aug 2007

15 Aug 2007

16 Aug 2007

17 Aug 2007

18 Aug 2007

Week 9

19 Aug 2007

20 Aug 2007

21 Aug 2007

22 Aug 2007

23 Aug 2007

24 Aug 2007

25 Aug 2007

Week 10

26 Aug 2007

27 Aug 2007

28 Aug 2007

29 Aug 2007

30 Aug 2007

31 Aug 2007

1 Sept 2007

Week 11

2 Sept 2007

3 Sept 2007

4 Sept 2007

5 Sept 2007

6 Sept 2007

7 Sept 2007

8 Sept 2007

Week 12

9 Sept 2007

10 Sept 2007

11 Sept 2007

12 Sept 2007

13 Sept 2007

14 Sept 2007

15 Sept 2007

Week 13

16 Sept 2007

17 Sept 2007

18 Sept 2007

19 Sept 2007

20 Sept 2007

21 Sept 2007

22 Sept 2007

Week 14

23 Sept 2007

24 Sept 2007

25 Sept 2007

26 Sept 2007

• Miniprepped the following
  • TetR-RBS-P2,21A-ter A,B,C and D
  • TetR-RBS-ComP-ter A,B,C and D
  • GenRBS-ComA-ter A,B,C and D
  • cI-L3 A,B,C and D

27 Sept 2007

28 Sept 2007

29 Sept 2007

Week 15

30 Sept 2007

1 Oct 2007

2 Oct 2007

3 Oct 2007

4 Oct 2007

5 Oct 2007

6 Oct 2007

Week 16

7 Oct 2007

8 Oct 2007

9 Oct 2007

10 Oct 2007

11 Oct 2007

12 Oct 2007

13 Oct 2007

Week 17

14 Oct 2007

15 Oct 2007

16 Oct 2007

17 Oct 2007

18 Oct 2007

19 Oct 2007

20 Oct 2007

Week 18

21 Oct 2007

22 Oct 2007

23 Oct 2007

24 Oct 2007

25 Oct 2007

26 Oct 2007