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Colony PCR analysis
Colony PCR failed: no bands


 * 171+174 (A-D):
 * lanes 1-4
 * expected size: 4615
 * good clones:
 * 171+175 (A-B):
 * lanes 5-6
 * expected size: 4416
 * good clones:
 * 170+175 (A-C):
 * lanes 7-9
 * expected size: 5265
 * good clones:
 * 170+174 (A-B):
 * lanes 10-11
 * expected size: 5464
 * good clones:
 * 170+173 (A-D):
 * lanes 12-15
 * expected size: 5471
 * good clones:

Transposase assembly
Conducted the following digestions: Gel image:

Self lysis test

 * started 2:00
 * checked at 4:30:

preparation of cells

 * scraped choanos and put 1ml in each test tube
 * added 40 uL bacteria to each test tube at 2:30
 * induced with arabinose fifteen minutes later (at 2:45)

Conor McClune 18:31, 12 October 2010 (EDT)

 * flow cytometry got no hits, because the choanos were in ASW, so the bacteria did not lyse

Self lysis test

 * started 2:15
 * checked at 4:15:

preparation of cells

 * scraped choanos and put 6ml in each test tube
 * added 240 uL bacteria to each test tube
 * induced with arabinose fifteen minutes later (at 2:30)

Plasmid Delivery Assay
Plasmid Payload Numbers:


 * 51 and 52 refer to the Self-lysis/Vacuole-buster devices iGEM10_051-052

Procedure
Choanos
 * aspirated two plates (15ml each) of choanos in ASW, resuspended each in 5mL of choano media
 * note: the choano media we planned on using was contaminated, so I had to use an older one (one that seemed to be lacking something because choanos have not grown very well in it)
 * scraped choanos, mixed the two plates (to get 10mL), aliquoted 500uL into each of 12 mini slide wells
 * added 20uL of bacteria to the wells (10:15am):
 * mistake:added 0.5uL ATC to each well at 10:30am, instead of arabinose (SLD is under Pbad not Ptet)
 * correction: added 5uL arabinose to each well at 11:00am

Self lysis test
 * made two 2mL aliquots of each of the plasmid bacteria cultures listed above
 * added 2uL ATC (mistake) and 20uL of arabinose to one of each of the aliquots of each culture

Results
Self Lysis Test
 * after 5 hours, all 10 plasmid+SLD/VB cultures with arabinose had lysed.

Eco/Bam transfer of iGEM10_171 (A) into Bca1256

 * transformed product into JTK159 and plated on spec

Re-Assembly of iGEM10_170

 * gel purified my previous digestions of iGEM10_159 and Bjh1906
 * ligated 4uL of each with 1uL t4 buffer and 1uL t4 ligase
 * transformed into JTK159 and plated on CA

LifeAct Assembly
Of the colonies if iGEM10_170 and iGEM10_171 picked yesterday, colonies 5,8,12 and 15 were cotransformed

Miniprepped: Colonies 1,2,3,7 (iGEM10_170) and colonies 10,11,18, 20 (iGEM10_171) because they were the reddest
 * renamed these clones iGEM10_170 A-D and iGEM10_171 A-D, respectively
 * iGEM10_171A sequenced well, with one mutation in the promoter
 * all of iGEM10_170 A-D are missing the terminator

LifeAct Assembly
There is growth on the plates I made yesterday for iGEM10_170-171, though the Red/green colonies on iGEM10_171 are very small

I picked 8 colonies for iGEM10_170 and 12 from iGEM10_171 (only the last 4 may be green AND red).

Ran a colony PCR, but it did not show much because the addition of the terminator is not visible on the gel. The gel for the last colonies


 * lanes 1-8: iGEM10_170
 * all but colony 8 look good
 * lanes 9-13: iGEM10_171
 * all look good, except that col PCR failed for colony 12

Restriction digest (Eco/Bam)of iGEM10_159-160

 * used iGEM10_159C
 * expected band sizes: 2660, 1580
 * the bands look good for both (faintess can be attributed to a bad zymo-forgot to spin out solvent for 90 sec before adding water)

Assembly of iGEM
Digested: Ligated each of the iGEM parts to left of Bjh1906 to make iGEM10_170-171, respectively Transformed into JTK159 cells and plated on CA
 * Xho/Bam: iGEM10_159C and iGEM10_160
 * Xho/Bgl: Bjh1906

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: today:
 * ligated the predigested combinations:
 * iGEM10_157 + Bjh1881
 * iGEM10_158 + Jtk2541
 * transformed both ligations into JTK159 strain
 * plated on CK

Results: no growth, failed 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



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.''

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

Lifact Assembly

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

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

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

Results:
 * note: broken well in lane 8, so contents were put in adjacent well
 * all lanes look good

Lifact Payload Assembly

 * Ran a gel of Tahoura's PCR of Pcon parts Bca1237 and Bca1238:
 * image: [[image:ConorGel100.jpg|100px]]
 * 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-

PCR magnification of Pcon part Bca1152
Gel Verification The Expand seems to have worked best. It has a strong band at 200bp
 * 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
 * expected size:206
 * gel purified the

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

PCR magnification of Pcon part Bca1152
Gel Verification
 * 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
 * expected size:1079

The band looks too long

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

PCR magnification of Pcon part Bca1152
Gel Verification
 * 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
 * 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

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

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

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.

Transposase Assay Results
No growth on any plates

PCR Isolation of Transposases
The following three pcr's were run on 2K55:

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.

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

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

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

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

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

Picking colonies for iGEM10_103 (iGEM10_006)

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