Berk2010-Conor

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To Do:

Things to try as payload (all from Josh):

• pcDNA3 - kan resistance (which should protect against G418) under eukaryotic promoter (sv40 promoter)
• pcmv promoter with RFP and GFP, on two different origins of replication (3 are amp, 2 kan, all ColE1 origin)

Conor McClune 19:59, 18 September 2010 (EDT)

LifeAct Assembly

Redigestion of iGEM10_156A and iGEM10_159D

(this is necessary because the previous version of

• Both were digested with Xho/Bam for 30 min
• Zymo'd
• Ligation (5:00-5:30):
  • iGEM10_156A + Bjh1881
  • iGEM10_159D + Bjh1906 (terminator)
  • iGEM10_159A + Bjh1906 (terminator)

Results: no growth for the iGEM10_159 + Bjh1906 assemblies, but one red/green colony for iGEM10_156A + Bjh1881 = iGEM10_160

• colony PCR of iGEM10_160:

• expected band : 1800bp, the band looks good

Remaking iGEM10_161 and iGEM10_162

(This is necessary because I had mislabelled iGEM10_157-158)
yesterday:

• ligated the predigested combinations:
  • iGEM10_157 + Bjh1881
  • iGEM10_158 + Jtk2541

today:

• transformed both ligations into JTK159 strain
• plated on CK

Results: no growth, failed assembly

Conor McClune 18:38, 17 September 2010 (EDT)

LifeAct Assembly

Restriction digest of iGEM10_159-162 with Eco/Bam

• expected sizes for all bands: 2660, 1580
• iGEM10_159: lanes 1-4
• iGEM10_160: lanes 5-8
• iGEM10_161: lanes 9-12
• iGEM10_162: lanes 13-16

Conor McClune 18:38, 14 September 2010 (EDT)

Assembly of LifeAct Payload

Potential Error discovered: plate with Eco/Bam iGEM157 and iGEM10158 were probably mislabelled since 157 was red and 158 was green (they should have been the green and red, respectively). This means that iGEM161-162 are probably just two copies of the same FP, one lifeact and one not.

Conor McClune 22:03, 12 September 2010 (EDT)

Lifeact Payload assembly

Digestion

• Lefty digested: iGEM10_155-158
• Righty digested: Bjh1881, Jtk2541
• incubated 3:30-4:20

Ligation

• ligating the following combinations:
  • iGEM10_155 + Jtk2541
  • iGEM10_156 + Bjh1881
  • iGEM10_157 + Bjh1881
  • iGEM10_158 + Jtk2541

Conor McClune 01:09, 10 September 2010 (EDT)

Lifact Assembly

• Eco/Bam'd iGEM10_155-158 into pMLL9 (CA) (for continued assembly) and also into 1256 (Spec) (for assaying in choanos)

Conor McClune 01:28, 9 September 2010 (EDT)

Lifact Payload Assembly

• I Eco/Bam digested iGEM10_155-158
• Gel purified smaller fragment of each
• meant to eco/bam into pMLL CA but we were out of the digested vector. I will have to digest this tomorrow

Conor McClune 18:04, 7 September 2010 (EDT)

Lifact Assembly

Colony PCR results

• colony # corresponds to lane #
• expected sizes:
  • lanes 1-4 Bca1237 + Bjh2252:1070
  • lanes 5-8 Bca1238 + Bjh2252:1070
  • lanes 9-4 Bca1237 + Bjh2251:1020
  • lanes 1-4 Bca1238 + Bjh2251:1020

• note: broken well in lane 8, so contents were put in adjacent well

Results:

• all lanes look good

Conor McClune 21:06, 5 September 2010 (EDT)

Lifact Payload Assembly

• Ran a gel of Tahoura's PCR of Pcon parts Bca1237 and Bca1238:
  • image:
    
  • cut out 200-300bp range for both parts and small fragment gel purified (eluted with 8.5uL H20)
• added 1uL each of NEB2, Bam and Eco to each of the purified products:
  • incubated at 37*C from 5:50-

Conor McClune 21:15, 13 August 2010 (EDT)

Assembly of iGEM10_146 and iGEM10_149

PCR magnification of Pcon part Bca1152

• template taken from Josh's plasmid stocks plate 5, well H8 *part is in plasmid pBca9145
• recipe: 50uL Phusion MM, 1uL ca998 10mM, 1uL g00101 10mM, 0.5ul template DNA
• run on 2K55

Gel Verification

• expected size:206
• gel purified the

The Expand seems to have worked best. It has a strong band at 200bp

Digestions

• for Bca1152 (Pcon)-recipe:
  • 8uL of eluted PCR product
  • 1uL of NEB Buffer 2
  • 1uL EcoRI
  • 1uL BamHI
• for Bjh2252(lifeact gfp) and Bjh2251 (lifeact rfp):
  • 14uL ddH2O
  • 2 uL NEB2+ATP Buffer
  • 1uL EcoRI
  • 1uL BamHI
  • 1uL miniprepped plasmid
• incubated for 1hr at 37
• zymo cleaned FP digests (eluted with 20uL), small fragment zymo cleaned Bca1152 (

Ligation

• recipe:
  • 10uL vector digest (Bjh2251 or Bjh2251)
  • 7.5uL Bca1152
  • 2uL Ligase
  • 2uL T4 buffer +ATP
• let sit on counter for 30 min

Transformation

• cell mixture: 100uL Jtk030, 15uL KCM
• 50uL of cell mixture added to each ligation mixture
• let sit on ice for a few minutes
• heat shocked at 42*C for 90 sec
• added 100uL 2yt
• rescued for 45 min
• plated both on CA

Conor McClune 14:40, 12 August 2010 (EDT)

PCR magnification of Pcon part Bca1152

• template taken from Josh's plasmid stocks plate 5, well H8 *part is in plasmid pBca1100
• recipe: 50uL Phusion MM, 1uL ca998 10mM, 1uL g00101 10mM, 0.5ul template DNA
• run on 2K55

Gel Verification

• expected size:1079

The band looks too long

Assembly of iGEM10_146 and iGEM10_149

Digestion

• standard Eco/Bgl digestion of Bjh2252 (GFP) and Bjh2251 (RFP)
• 11:50-1:00
• product was zymo purified

Ligation

• recipe: 3uL H20, 1uL T4 buffer, 1uL T4 ligase, 1uL FP part (Bjh2252 or Bjh2251), 4uL yesterdays Eco/Bam digest of Expand PCR of Bca1152
• incubated on desk from 11:50-12:30

Transformation

• cell mixture: 200uL JTK030 cells, 30uL KCM
• added 70uL of this mixture to each of the ligation mixtures
• heat shocked for 2 min
• added 100ul 2YT
• rescued for 1.5 hours
• plated 100ul of iGEM10_146(lifact ffGFP) assembly on CA
• plated 100ul of iGEM10_149(lifact RFP) assembly on CA

Picking colonies of iGEM10_145

• picked 4 green fluorescent colonies
• ran a colony PCR with ca998 and G00101
  • expected size: 964

• no visible bands

Conor McClune 17:30, 11 August 2010 (EDT)

PCR magnification of Pcon part Bca1152

• template taken from Josh's plasmid stocks plate 5, well H8 *part is in plasmid pBca1100
• conducted PCR with both Phusion and Expand master mixes
• recipe: 50uL MM (phusion or expand), 1uL ca998 10mM, 1uL g00101 10mM, 0.5ul template DNA
• both run on 2K55

Gel Verification

• expected size:1079
• row 1 : 10uL phusion product
• row 2 : 10uL expand product

• both products are the correct size, though yield is weak
• Phusion product looks best

Assembly of Lifact Payload Parts iGEM10_144-145

Digestion

• recipe: 4uL H20, 4uL DNA, 1uL NEB2, 1uL enzyme 1, 1uL enzyme 2
• digested Bjh1883 and jtk2541 with Eco/Bgl
• digested both phusion and expand PCR products of Bca1152 with Eco/Bam
• digested 4:53-

Zymo Cleanup

• conducted a standard zymo cleanup of Bjh1883 and jtk2541
• conducted a small-fragment zymo cleanup of PCR products

Transformation

• cell mixture: 200uL JTK030 cells, 30uL KCM
• added 70uL of this mixture to each of the ligation mixtures
• heat shocked for 2 min
• added 100ul 2YT
• rescued for 2.5 hours
• plated 100ul of iGEM10_145(ffGFP) assembly on Spec
• plated 100ul of iGEM10_144(RFP) assembly on CA
  • since we were out of CA plates, I diluted 25uL 1000X Amp with 75uL H20 and plated this onto a Chlor plate

Conor McClune 15:07, 10 August 2010 (EDT)

Testing the SLD in our transposase parts iGEM10_007,13,20

Last night I picked colonies of each and grew them up in 6ml LB. iGEM10_007(piggybac) did not grow up because it was in the wrong color LB, but I repicked it at 11:30 and will test it later.

iGEM10_013(sleeping beauty) and iGEM10_020(Tn5):

• split each culture into two aliquots of 3mL - one with 3uL atc, one with nothing added
• placed these cultures on the incubator at 12:05

results at 1:05: no lysis

results at 2:05: no lysis

Conor McClune 17:19, 9 August 2010 (EDT)

Self lysis in Varied Media:Results

Procedure:

• Grew up 10mL of two colonies off the plates for igem10_020, 013, and 131
• Used 8mL of those cells by filling 4 collection tubes with 2mL of cells. Spun them down and disposed of supernatant.
• Resuspended one of each in TB, LB, Sea water, and Transposase Buffer.
• Divided each of the 12 2mL sample into two wells on a 24 well plate.
• Added 1uL of atc to one of the 1mL samples from each 2mL sample. See detailed layout of plate above.
• Moved plate to 37deg shaker. Let sit for 1hour.
• Transfered 200uL of cells from each well to an eppendorph tube and spun down the cells. Transfered supernatant to Zymo Columns.
• Performed a zymo. Note: used 800ul of ADB to meet the necessary 1:4 ratio. Eluted with 10uL.
• Put tubes on ice. Transformed a huge batch of Righty cells (pir +). Added 70uL of cells to each tube.
• Heat shocked, added 100uL of 2YT, let rescue for ~10min before realizing the plasmids are AC and I could just plate on Amp.
• Plates are currently stored in incubator labeled 1-24 according to the layout above.

'	iGEM10_20 (atc)	iGEM10_20 (no atc)	iGEM10_013 (atc)	iGEM10_013 (no atc)	iGEM10_131 (atc)	iGEM10_131 (no atc)
Transposase Buffer	32	5	7	8	very high density	high density
ASW	70	135	35	49	high density	higher density
LB	490	146	119	105	high density (almost lawn)	high density (almost lawn)
TB	234	158	73	122	0	high density (almost lawn)

Conor McClune 14:31, 5 August 2010 (EDT)

Transposase Assay Results

No growth on any plates

His-tag addition to Transposases

PCR Isolation of Transposases

The following three pcr's were run on 2K55:

Well	Name	Vector	Target Part	Fwd Oligo	Rev Oligo
1	sbb10	KA	<SB100x!	ca998	igemTen048
2	sbb04	KA	<piggyBac!	ca998	igemTen050
3	iGEM10_055	KA	<tn5>{<NIS!}	ca998	igemTen051

Conor McClune 14:30, 4 August 2010 (EDT)

Transposase Assay: Testing Sleeping Beauty and Tn5 in NEB2+MgCl2, varying [DNA] and activity time

Transposition Buffer: Added 1.0165mg to each mL of NEB2 (MgCl2 tetrahydrate is 203.3g/mol and we're attempting to copy a transposition buffer that contains 150mM of MgCl2). We added 36.6mg of MgCl2 to 32.4mL of water and 3.6mL of 10xNEB2 to make 150mM MgCl2 NEB2. We also added 5 drops of diluted HCl (tube labeled diluted HCl... we made it my dipping a pasteur pipette in the 12M HCL and then in 50mL of mgH20) to bring the pH from 7.8 to 7.6 or so.

Spun down all 5mLs of cells and 1uL of control cells (no ara). Resuspended in NEB2+MgCl2 buffer. Transferred control into a tube. Combined all 5mL resuspended cells into one tube, and then separated into 5 tubes.

Added 1uL of atc and 10uL of arabinose to each mL of cells at 1:05pm. Put in 37deg shaker.

Started adding DNA at 1:40pm. For DNA, we mixed two minipreps of 9145-1144. Well, we mixed them after adding DNA from one of the MPs to the 1uL tubes and the 13 .5uL tube (Whoops).

Somehow, we ran out of MP, so we're only adding up to 10uL of DNA.

Time points:
.5hr- 2:10pm
1hr- 2:40pm
1.5hr- 3:10pm
2hr- 3:40pm

Transform into TG1 cells. We mixed 11 tubes of TG1 cells together in a Falcon tube.

Conor McClune 16:53, 3 August 2010 (EDT)

Assembly of iGEM10_007

The assembly of this part is being put on pause until it is confirmed that the identical part iGEM10_131 is in fact incorrect. The plate with colonies is in the refrigerator.

Analytical PCR of iGEM10_131

Well Contents: 33uL Taq MM, 1uL each oligo, 0.5uL template DNA

• run on 4K45

Well	Part	Fwd Oligo	Rev Oligo	Expected Size	Acceptable?
1	iGEM10_103	ca56	igemTen004	2045	yes
2	iGEM10_103	ca56	igemTen006	2411	yes
3	iGEM10_103	ca56	igemTen008	3475	no

Preparation of Bacterial Stock Choano Food

• Grew up 400mL MC1061 overnight on 37*C shaker
• split into two centrifuge bottles and centrifuged at 4200rpm for 14 minutes
• poured off supernatant
• washed each pellet with 10mL artificial sea water (ASW)
• poured off ASW
• added 10mL ASW to each bottle and resuspended pellets
• centrifuged at 4200rpm for 14 minutes
• poured off supernatant
• resuspended pellets
• combined the contents of the two bottles
• created 2uL aliquots

Conor McClune 14:39, 2 August 2010 (EDT)

Miniprep of iGEM10_103

The bacterial growth was smaller than usual, so I eluted with only 25uL H20, in order to avoid having a small concentration of DNA. I miniprepped all 6 picked clones of iGEM10_103.

Restriction Mapping

• Digested 4uL of each miniprep with Xho/Bgl. Incubated from 11:30-12:15. Froze from 12:15-1:15.
• expected fragments: 6995 (desired fragment), 1607 (vector fragment)

• no bands visible
• I want to gel purify the larger of the two fragments, so I cut out the entire 7kb section of all six rows, hoping to capture even a low concentration of the correct piece.

Analytical PCR

Well Contents: 33uL Taq MM, 1uL each oligo, 0.5uL template DNA

• run on 4K45

Well	Part	Fwd Oligo	Rev Oligo	Expected Size	Acceptable Clones
1	iGEM10_103	ca998	igemTen004	1859	1-3,5,6
2	iGEM10_103	ca998	igemTen006	2225	1-3,5,6
3	iGEM10_103	ca998	igemTen008	3289	none

Gel Pictures

• ca998 + igemTen004

• ca998 + igemTen006 (lanes 1-6) and ca998 + igemTen008 (lanes 7-12)

Results
Either a part is missing around the 3'TerminalRepeat, or the pcr is bad. I will repeat it tomorrow

Assembly of iGEM10_007

Digestion

• I used a stored, previously digested version of iGEM10_001
• I used the above Xho/Bgl digestion of iGEM10_103 (aka iGEM10_006)

Ligation

• Recipe: 7uL iGEM10_103 purified digest, 1uL iGEM10_001 purified digest, 1uL T4 Buffer, 1uL T4 ligase
• incubated on desk from 2:55-3:25

Transformation

• cell mixture: 100uL JTK030 cells, 50uL H20, 30uL KCM
• added 70uL of this cell mixture to the ligation mixture
• heat shocked for 90sec
• added 100uL 2YT and rescued for 1 hr

Transformed iGEM10_013 #5

• transformed into JTK030 cells for use in a transposase assay
• cell mixture: 100uL JTK030 cells, 50uL H20, 30uL KCM
• added 70uL of this cell mixture to the ligation mixture
• heat shocked for 90sec
• added 100uL 2YT and rescued for 1 hr

Grew up MC1061 cells to feed choanos in ASW

4:00pm:

• added 100uL MC1061 competent cells to 400mL LB
• placed on shaker at 37*C overnight

Conor McClune 23:44, 1 August 2010 (EDT)

Picking colonies for iGEM10_103 (iGEM10_006)

• Picked 6 colonies - grew up in KA LB
• Colony PCR
  • primers: igemTen004 and ca56