Berk2010-Christoph

Christoph Neyer 19:04, 11 June 2010 (EDT)

Next step is to transform each part into a Righty or Lefty strain.
Manually transformed parts from dilution plate into Righty and Lefty strains. Incubated at 5:10. Take out at 5:50.
Transformation plate has same layout as dilution plate below. Here is layout again:

'

1

2

3

4

5

6

7

8

9

10

11

12

A

6+2

6+11

6+22

7+9

7+24

5+23

8+17

4+10

9+3

9+20

7+24

B

6+2

6+11

6+22

7+10

5+3

5+23

8+17

4+10

9+3

9+20

9+11

C

6+4

6+12

6+23

7+11

5+3

8+1

8+18

4+16

9+8

9+23

9+23

D

6+4

6+12

6+23

7+13

5+8

8+1

8+18

4+16

9+8

9+23

pMLL5+Bca1091 (L)

E

6+5

6+13

6+24

7+14

5+8

8+4

8+21

4+17

9+11

pMLL4+Bjh1882 (L)

F

6+5

6+13

6+24

7+19

5+15

8+4

8+21

4+17

9+11

pMLL6+Bjh2245

G

6+7

6+19

7+2

7+23

5+20

8+6

4+9

4+18

9+13

6 +24

6+12

H

6+7

6+19

7+5

7+24

5+20

8+6

4+9

4+18

9+13

7 + 23

5+25 (D)

Christoph Neyer 16:31, 11 June 2010 (EDT)

Did a test run with the robot using our dilution csv file. Checked cloumn 4 it worked well.
Miniprepped pMLL5+Bca1152 and sent in for sequencing as iGem021.
Using robot to dilute 4ul of each vector+part stage 0 assembly into 76ul of water. See this csv file: Media:Stage0PartsDilution.csv

Source plate layout:

'

1

2

3

4

5

6

7

8

9

10

11

12

A

6+2

6+11

6+22

7+9

7+24

5+23

8+17

4+10

9+3

9+20

7+24

B

6+2

6+11

6+22

7+10

5+3

5+23

8+17

4+10

9+3

9+20

9+11

C

6+4

6+12

6+23

7+11

5+3

8+1

8+18

4+16

9+8

9+23

9+23

D

6+4

6+12

6+23

7+13

5+8

8+1

8+18

4+16

9+8

9+23

pMLL5+Bca1091 (L)

E

6+5

6+13

6+24

7+14

5+8

8+4

8+21

4+17

9+11

pMLL4+Bjh1882 (L)

F

6+5

6+13

6+24

7+19

5+15

8+4

8+21

4+17

9+11

pMLL6+Bjh2245

G

6+7

6+19

7+2

7+23

5+20

8+6

4+9

4+18

9+13

6 +24

6+12

H

6+7

6+19

7+5

7+24

5+20

8+6

4+9

4+18

9+13

7 + 23

5+25 (D)

Destination plate layout:

Columns 1-6 need to be transformed into Righty strains. Columns 7-12 need to be transformed into Lefty strains.

'

1

2

3

4

5

6

7

8

9

10

11

12

A

4 with 16

5 with 20

6 with 12

7 with 10

8 with 17

9 with 11

4 with 10

6 with 11

6 with 5

7 with 13

8 with 1

9 with 3

B

4 with 17

5 with 23

6 with 13

7 with 11

8 with 18

9 with 13

4 with 18

6 with 12

6 with 7

7 with 14

8 with 21

C

4 with Bjh1882

5 with 25

6 with 22

8 with 4

9 with 20

4 with 9

6 with 19

6 with Bjh2245

7 with 19

8 with 6

D

5 with 3

6 with 23

9 with 23

6 with 2

7 with 2

E

5 with 8

6 with 7

9 with 8

6 with 22

7 with 23

F

6 with Bca1091

6 with 23

7 with 24

G

5 with 15

6 with 24

7 with 5

H

5 with 3

6 with 4

7 with 9

Christoph Neyer 13:57, 11 June 2010 (EDT)

• Added grown up Lefty and Righty strains to 500ml each of LB. Growing up for 2-3hrs. Check at 1pm.
• Running colony PCR gel for Bca1152+pMLL5. Expected band at ~260.

Colonies A,C,D look good. Chose C and D to miniprep. Cells were put in warm room at 9am.
Grab Bca1152 colony PCR colonies at 1 or 2pm.

• Added missing parts to iGEM10 plate1 minis:

'

1

2

3

4

5

6

7

8

9

10

11

12

A

6+2

6+11

6+22

7+9

7+24

5+23

8+17

4+10

9+3

9+20

7+24

Lysis Device

B

6+2

6+11

6+22

7+10

5+3

5+23

8+17

4+10

9+3

9+20

9+11

5+25

C

6+4

6+12

6+23

7+11

5+3

8+1

8+18

4+16

9+8

9+23

9+23

D

6+4

6+12

6+23

7+13

5+8

8+1

8+18

4+16

9+8

9+23

pMLL5+Bca1091 (L)

E

6+5

6+13

6+24

7+14

5+8

8+4

8+21

4+17

9+11

pMLL4+Bjh1882 (L)

F

6+5

6+13

6+24

7+19

5+15

8+4

8+21

4+17

9+11

pMLL6+Bjh2245

G

6+7

6+19

7+2

7+23

5+20

8+6

4+9

4+18

9+13

6 +24

6+12

H

6+7

6+19

7+5

7+24

5+20

8+6

4+9

4+18

9+13

7 + 23

Christoph Neyer 21:03, 10 June 2010 (EDT)

TO DO:

• Pick colonies from Bca1152 and do colony PCR. Then miniprep if time.
• Play with robot
• Make competent cells.
• Transfer box parts to dilutions plate.
• When oligos arrive. Attempt biobrick on Bjh2342CA.

Christoph Neyer 13:38, 10 June 2010 (EDT)

• Had to re-grow Lefty and Righty strains since no colonies grew.
• Need to make part 5 with 25 (pMLL5-CK+B10sbb37) or (pMLL5-CK+Bca1152)
  

Currently in DH10B. Need to Eco/Bam transfer it into pMLL5-CK.
Put E/B digest of Bca1152 in DH10B into thermocycler at 12:10pm. Take out at 1:10pm and do small fragment zymo cleanup.
Set up ligation of Bca1152 and pMLL5 at 2:15. Transform at 2:45.
Heat-shock transformed Bca1152+pMLL5 into Jtk049. Plate at 4:15pm.
Plated Bca1152+pMLL5 into Jtk049. Growing up over night.
Sent in iGEM020 for sequencing(6 with 12C from working box to double check).

Christoph Neyer 20:31, 9 June 2010 (EDT)

TO DO List:

• Finish generating competent cells for "Lefty" and "Righty" strains.
• Analyze sequencing data for pMLL6+Bjh2245 B and C.
• Transform pMLL6+Bjh2245 into "Righty" strain.
• Transform other parts into the correct "Righty" or "Lefty" strains to prepare for robot 2ab assembly.

Christoph Neyer 15:16, 9 June 2010 (EDT)

Colony PCR of pMLL6+Bjh2245 should be at about 265bps. Looks good. Wait for colonies to grow and then miniprep.

• Sent in miniprepped pMLL4-Bjh1882 (D) and pMLL5-Bca1091 (2A) for sequencing.
• Started growing up Lefty and Righty strains. Picked two colonies of each lefty and righty and grew them up in 10ml LB.
• Designed three potential oligos for biobricking part Bjh2341CA (igemTen011, igemTen012, and igemTen013)
• Miniprepped pMLL6+Bjh2245. Need to transform into "Righty strain"
• Sent in minprepped pMLL6+Bjh2245 to be sequenced.

Christoph Neyer 13:28, 9 June 2010 (EDT)

Bca1091 is 60bps.
Colony PCR band for parts:

• pMLL4+bjh1882 = 849bps
• pMLL6+bjh2245 = 265bps
• pMLL5+bca1091 = 228bps

• For First try Colony PCR of 1882 and 1091:
  • Bca1091 pick lanes 8 and 10 for miniprepping.(Block #1: Colonies from well 1D and 2A)

• For Second try Colony PCR of 1882 and 1091
  • Bjh1882 pick lanes 1 and 4 for miniprepping. (Block #2: Colonies from well A and D)

Christoph Neyer 19:37, 8 June 2010 (EDT)

TO DO Tomorrow:

• Miniprep colonies that we pick for pMLL4+Bjh1882 and pMLL5+Bca1091. These are methylated correctly for the robot.
• Colony PCR pMLL6+Bjh2245 in Jtk049. These are not methylated correctly.

Christoph Neyer 19:51, 8 June 2010 (EDT)

• Ran gel of colony PCR pMLL4-Bjh1882 and pMll5-Bca1091 AGAIN:

ladder

1882a

b

c

d

1901a(1)

b

c

d

ladder

1901a(2)

b

c

d

Christoph Neyer 16:59, 8 June 2010 (EDT)

• Ligated pMLL6 Eco/Bam digest with part Bjh2245.
• Ran gel of colony PCR pMLL4-Bjh1882 and pMll5-Bca1091:

ladder

1882a

b

c

d

1901a(1)

b

c

d

ladder

1901a(2)

b

c

d

• Repicking colonies since Colony PCR appeared contaminated. Block has same layout, shown below:

PCR Block 1882 and 1091 #2	'	'	'	'
1882		1091 #1		1091 #2
A		A		A
B		B		B
C		C		C
D		D		D

• Ran out of "Righty" cells. So we are transforming Jtk049 with our E/B ligation of pMLL6+Bjh2245.

Christoph Neyer 14:04, 8 June 2010 (EDT)

• Digested pMLL6 w/ Eco/Bam and gel purified.
• Colony PCR of transformed pMLL4-Bjh1882 and pMLL5-Bca1091:
  

PCR Block 1882 and 1091 #1	'	'	'	'
1882		1091 #1		1091 #2
A		A		A
B		B		B
C		C		C
D		D		D

Christoph Neyer 19:30, 7 June 2010 (EDT)

Bjh1882 Vectors: AC
Bjh2245 Vectors: KA
Bca1091 Vectors: CK
pMLL4-AC
pMLL5-CK
pMLL6-KA
pMLL7-AK
pMLL8-KC
pMLL9-CA
Bjh1882+pMLL4-AC = Lefty
Bjh2245+pMLL6-KA = Righty
Bca1091+pMLL5-CK = Lefty
Ligated isolated Bjh1882 and Bca1091 into above vectors. Transformed into Lefty strains.
Need to ligate Bjh2245 (Missing pMLL6, need to digest).
TO DO:

• Digest pMLL6 with Eco/Bam and gel purify. Then ligate with Bjh2245.
  
• Check transformed Bjh1882 and Bca1091.
• Transform ligated Bjh2245+pMLL6 into Righty strain

Christoph Neyer 16:35, 7 June 2010 (EDT)

Couldn't see fragments. Turns out Bjh2245 is 97bp and Bjh1882 is ~680bps.
Re-cut Bjh2245 and Bjh1882.
Small fragment zymo cleanup of Bjh2245 digest.


Gel purified Bjh1882.

Christoph Neyer 15:54, 7 June 2010 (EDT)

Eco/Bam digest of parts: Bjh2245 (LifeACT), Bjh1882 (RFP), and BCA1091 (Ptet).
Gel purified Bjh1882 and Bjh2245.
Small fragment zymo cleanup of BCA1091.