Berk2010-Amy
To Do
On Thursday:
- Map and submit for sequencing: 1884+1858 in jc012 samples, 41/42+43/44 samples
- If oligos come in, PCR off transposases
If time:
- Look up crude extract buffers for different transposases
- Electroporate purified transposase after exposing it to DNA?
- Plan self-lysis assay (transposase buffers-NEB,PBS+MgCl2, LB, Choano Media, Choano media+TB)
- Plan Tecan assay?
- Tecan: compare self-lysis device with L vs. T lysozyme, in TB/choano media/LB/1mlchoano
Background stuff:
- look up transposase expression times
- reverse transcriptase--deliver rRNA
- integrase... needs integration sites
Amy N. Kristofferson 14:45, 6 August 2010 (EDT)
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
Amy N. Kristofferson 13:28, 5 August 2010 (EDT)Yesterday's Assay failed. No colonies grew. We're going to do a self-lysis assay tomorrow to trouble shoot. Picked two colonies of iGEM20, 13, and 131 and inoculated in 10ml LB-AC. Also, streaked each on Gen, just to make sure GenR cassette is functioning. Miniprep Mapping: 1884+1858 in jc012 and 41/42+43/44 samples1884+1858 in jc012: Eco/Bam should give 712 and 5713bp. igem10_045 Eco/Bam: 5548, Eco/Bam digest started at 3:46pm. I ran the gel too long, but I'm going to submit the first one for sequencing. Hopefully I have oligos I can use... Minipreps of 9145-1144Made 8 and put them in Amy #2. Amy N. Kristofferson 13:53, 4 August 2010 (EDT)MiniprepsNone of the 1884/1858 samples were cotransformed, but out the the 41/42+43/44 samples, 41+44b and 42+44a were cotransformed. I miniprepped everything else.
Transposase Assay: Testing Sleeping Beauty and Tn5 in NEB2+MgCl2, varying [DNA] and activity timeTransposition 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: Transform into TG1 cells. We mixed 11 tubes of TG1 cells together in a Falcon tube. Self Lysis Test in NEB2+MgCl2With bacteria grown up from another colony for Transposase Assay (Arabinose added last night around 5:30pm), with took 2mL, spun then down and resuspended in our NEB2+MgCl2 buffer. I divided the 2mL into 2 tubes and added 10uL of arabinose (just to keep it similar to the above experiment) and 1uL of atc to one of them. We'll let it sit in the shaker for 1 hr, and then we'll spin down the cells, zymo the supernatant, transform jtk030 (pir+) cells and them plate on Gen. 1:24pm. Transposase PurificationLiterature search:
Tn5: N-terminus (1)
(2)http://www.nature.com/mt/journal/v15/n1/pdf/6300028a.pdf "We added a hemagglutinin (HA) epitope tag to the N-terminus of the piggyBac transposase and demonstrated no effect on transposition activity compared with the native enzyme (data not shown)."
For a typical reaction, 2 μl (approximately 0.2 μg of protein/μl) of Tnp was added to 18 μl of pRZTL1 plasmid (approximately 1 μg of DNA) in the transposition reaction buffer (0.1 M potassium glutamate, 25 mM Tris acetate, pH 7.5, 10 mM Mg2+acetate, 50 μg/ml bovine serum albumin, 0.5 mMβ-mercaptoethanol, 2 mM spermidine, 100 μg/ml tRNA; final concentrations). The reaction was incubated for 1 h at 20 °C and then diluted 2–3-fold in the same buffer and transferred to 37 °C. This procedure was performed to facilitate binding in the presence of CHAPS present in the reaction mixture as a component of the Tnp storage buffer (TEGX, 10 mM CHAPS). Dilution increased the cleavage reaction presumably due to dilution of CHAPS. CHAPS can be eliminated from the reaction (and storage buffer), and a simple incubation at 37 °C with no dilution is satisfactory if Qiagen-purified DNA is used. We nevertheless used the above two-step procedure for all experiments to synchronize the start of cleavage. (from "Tn5 in Vitro Transposition" http://www.jbc.org/content/273/13/7367.full)
design oligo to add xhoI site immediately after self-lysis device. PCR off igem 19, 103, 104 (sequence 103 and 104 first?) w/ ca998 and above oligo. Bgl/XhoI PCR product into Mike's vector. igem10_019: <tn5>{<NIS!}b1006Tn5 5'TRPromoter_rbs_GenRTn5 3'TRSelf_Lysis_Device
Sleeping Beauty may contain an internal NLS: http://www.nature.com/mt/journal/v9/n2/pdf/mt200424a.pdf Amy N. Kristofferson 19:09, 3 August 2010 (EDT)I picked four colonies of each of the 41/42 and 43/44 combinations (a good number of colonies grew up on all of the plates) and four colonies of the restreaked 1884+1858 in jc012 vector. I checked them all for cotransformation, but skipped the colPCR since the parts are so large and jco12 doesn't have ca998/goo101. Conor and I also picked colonies in order to set up for the Transposase Assay tomorrow. We're going to perform the assay in the following buffer's: Transposition Reaction Buffer 10×transposition buffer (100 mM Tris, pH 7.5, 150 mM MgCl2, 100 mM KCl, 10 mM DTT) NEB2 1X
10 mM Tris-HCl, 10 mM MgCl2, 50 mM NaCl, 1 mM DTT (pH 7.9 at 25°C) Phosphate Buffered Saline
10X Solution
1.37M Sodium Chloride, 0.027M Potassium Chloride, and 0.119M Phosphate Buffer Oligos ordered: igemTen050 gctagCTCGAGttaGAAGCAAGACTGGCACATG 33 rev xhoI piggybac For Tn5, I can use the oligo I designed to go off the NLS: Here are the PCR reactions I'll have to set up: Amy N. Kristofferson 14:30, 2 August 2010 (EDT)To do:
Results from Tn5 Toxicity AssayNotes for Transposase Assaypbad-transposase-tr-gen-tr-self lysis (no zf) tn5: igem10_020, igem10_117 piggybac: igem10_131 sleeping beauty: igem10_134 (not made yet) Streaking dilution of 1884+1858 in pjc012I'll pick colonies tomorrow, miniprep and sequence on Wednesday. Map of 46 and 49Started Eco/Bam digest at 11:29am. Lane 1-3: 46 c,d,g Expected lengths: 6117, 2877 46d and g look good. None of 49 look good. I submitted 46d and 49a for sequencing. Redo Assembly of 45 and 50Redo of digests, using NEB3 buffer instead. 42+43=45 41+44=40 Bgl/XhoI digest of 43 and 44 Bam/XhoI digest of 41 and 42 Started at 12:12pm. Since my minipreps are all over the map, I'm going to redo all combinations of 41/42+43/44. Ligation started at 2:08pm. Started rescue at 3:22pm. Amy N. Kristofferson 02:05, 2 August 2010 (EDT)1884+1858 in jc012 restreakingSince these plates made lawns with only a few tiny green colonies, I went back and swabbed the area of the plate with a green colonies and dipped it in 100uL of 2YT and then plated all the 2YT on Amp. Hopefully this will give me larger, isolated green colonies. Amy N. Kristofferson 14:21, 29 July 2010 (EDT)To do:
iGEM10_046 and 051 Assembly analysisColPCR lanes 1-8: 42+44 (iGEM10_046) expected length: 6317bp Perfect: 4,7 MP: 4,7 lanes 9-12: 41+43 (iGEM10_049) expected length: 5978bp Perfect: 4 MP: 1,2 (4 was cotransformed) Cotransformation results: Only 41+43d was cotransformed. Unfortunately, d was the only one that looked good on the colPCR. Oh well. Miniprepped: Tecan Assay: Testing Self-Lysis timing of 047/048 in LB and Toxicity of 020Assembly of 1888+1858 with Mike's plasmidMessed up yesterday's assembly, and realized I don't need to gel purify the digest of Mike's plasmid, because I'll be able to select for the correct plasmids, since the colonies will be green So I set up the following: Bgl/XhoI digest of 1884+1858 e,f eluted PCR products Bam/XhoI digest of Mike's plasmid (mel001 in pJco12) Started at 12:55pm. Ligation started at 2:20pm. Transformed jtk030 cells and plated on amp. Amy N. Kristofferson 14:10, 28 July 2010 (EDT)To do:
41/42+43/44 plates Analysis and colony picking42+44: 18 colonies I'll pick 8 colonies off 42+44 (I have a feeling a lot of them are co-transformed), and 4 colonies off 41+43. Inoculate in LB-AK, colPCR?, streak on triple antibiotics. Ran a 7KcolPCR Restock 9145-1144Transformed jtk030 cells. 1884+1858 ca998/iGEM048Expected length: ~700bp. They all look great! I'll also digest Mike's plasmid (Mel001 p3co12) with Bam/XhoI. Started at 2:14pm. Even though I haven't gotten sequencing results back, I'm going to digest sample c and d with Bgl/XhoI. Digest started at 3:06pm. Gel purified. Expected lengths: 700bp dropout, vector should be ~5700bp. (Single cut would produce ~6400bp) Hopefully the gp worked well... I cut out the upper band, which was too high to really distinguish the length.
Transformed jtk030 cells. Found my digests in the incubator. Turns out I used the undigested eluted PCR product for the assembly. Whoops. Trashed the cells. I'll redo the digestion+ligation tomorrow. Transposase Assay: Testing Transposase Expression time and Activity Time
Need to transform cells w/ 9145-1144 so we can get more DNA. Transformed TG1 cells. Rough test of Self Lysis Timing in TBYesterday, I inoculated iGEM10_047 and 048. I put 1 mL of each into two tubes, so I have a control tube and a tube to add atc to, with all the cells derived from one colony pick. Added 1uL of Conor's 1000x atc at 11:25am. At 12:25pm, the cells still do not appear to have lysed. Check cells at 11:55am. Based on visual inspection, there appears to be no change in OD. Checked cells again at 1:00pm. The cells in 47 have clearly lysed, but it doesn't appear that the 48 cells have lysed yet. Pictures: 47 (w/o tip) Left=control, right=atc 48 (w/ tip) left=control, right=atc. Sequencing ResultsiGEM10_042a is perfect. (ca998 sequencing redo produced a perfect partial) Degradation AssayConor will do this tomorrow: Add 1uL of atc to each 1mL sample of cells. Let cells lyse for an hour. Add 1uL of DNA. At each of the following intervals: 15min, 30min, 45min, 1 hour, 1.5, 2, 2.5, 3, remove 200uL of DNA, spin down, and zymo supernatant. After you have all your zymo'd samples, plate on Amp. Amy N. Kristofferson 14:25, 27 July 2010 (EDT)To do:
PCR off 1884+1858 in 1601CKI designed an oligo that will add a XhoI site to the NLS. So if I do a PCR with ca998 and my oligo igemTen048, hopefully I should get a band around 733bp long. I should only get this band if NLS is after RFP. It can't PCR off just RFP, because there would be no NLS to bind to. And just NLS is only 33bp, so that wouldn't even show up on the gel. Ran a 2K55. Mapping of 1884 and 1858I wasn't going to map these, but since sequencing of 1884+1858 a and b w/ g00101 both failed, I'm going to map them. The maps won't show whether or not the NLS tag is there, but it should show how good the MP is and if the backbone is good. Eco/Bam Digest started at: 12:30pm Expected lengths: 3142 (vector), 711 (part) Lanes 1-8 correspond with samples a-h. Everything looks good. They could have had bad reads because they were cotransformed. I submitted 1884+1858c and d for sequencing. Sequencing ResultsiGEM10_020b in pmllCA is perfect Next Assembly step: Creating iGEM10_045/046 and 049/050 (Self-Lysis T ver)Assemble iGEM10_042/041 with iGEM10_43/044 I have left over righty digest of iGEM10_043/044, so I only have to set up a Lefty digest of iGEM10_042/041. NOTE: iGEM10_042 hasn't been sequence confirmed yet, but I'm going to go ahead with assembly and keep my fingers crossed. I think I started it around 2:00pm. Started ligation at 4:37pm. Used 2114+2275/2316 digests from construction of 47 and 48. Amy N. Kristofferson 14:07, 26 July 2010 (EDT)To do:
OligosDesigned the following oligo: igemTen048 gctagCTCGAGttaAACTTTACGTTTTTTTTTCGGCGG 38 rev xhoI 1858 {<NLS!} ColPCR resultsExpected length: about 900bp. They all look good. Mapping won't show me much, since the NLS is so small (~30bp). So I'm going to sumbit two samples for sequencing, using the g00101 oligo. If they're cotransformed, I'll get back a mixed read. Expected lengths for iGEM10_042: 2895bp (lanes 1-4) and iGEM10_041: 3126bp (lanes 5-8) Results: 42a,b,c and 41b,d look good. I'll map all the minipreps anyway. Miniprep MappingThey all look good! iGEM10_020 is a little faint, but I'm going to send off a sample of each to be sequenced.
started at 12:00pm. Transformed jtk030 with iGEM10_020b=Started rescue at 2:25pm. Labeled "Sequenced MP of iGEM10_020b." I'll grow up colonies tomorrow so I can perform the Transposase Assay on Wednesday. Sequencing submissionsSubmitted iGEM10_020b in pmllCA, iGEM10_042a in pmllAC, iGEM10_041b in pmllCA, and jh1884+1858 in 1601CK A,B for sequencing. Bam/XhoI digest started at 1:50pm. Amy N. Kristofferson 14:07, 24 July 2010 (EDT)To do:
Colony picking: 42/41PCR+pMLL4AC and iGEM10_020Picked 8 colonies of iGEM10_020 and checked for cotransformation. Didn't run a colPCR b/c the part is too big. Picked 4 colonies of 42/41PCR+pMLL4AC and started a 4K colPCR. Had to make more colPCR MM.
Miniprepped all 1884+1858 samplesLabelled 1884+1858a-h and placed in Amy #2. I need to map them. Sequencing Results for iGEM10_019bThe part looks great! I'm glad I continued on with assembly.
Amy N. Kristofferson 13:45, 23 July 2010 (EDT)To do:
Later...
Colony Picks1884+1858: expected colPCR length: 912bp efla/atub+2093 expected colPCR lengths: 1952, 2190bp Ran col3k pcr.
Assembly: iGEM10_020 and Eco/Bam 41 and 42 PCR products into pMLL4ACDigests: eco/bam: 41, 42 R: bgl/xhoI of igem019-gel purify (mapping at same time): 6181, 1607. Cut out top band. L:bam/xhoI of igem001
Gel purified 19b,d: There was a band at around 6000bp, but I couldn't see one at 1607, but that could be because I ran the gel for a long time. I gel purified the top band anyway and I'll continue on with the next step of assembly. I could map again, but I think my analytical colPCR combine with sequencing results will be a pretty good confirmation. Zymo'd 41, 42, and 1. Ligations: 41/42+pMLL4AC, transform into lefty 19b/d with 1, transform into jtk030
Rescue started at 3:41pm. PCR of ca998/g00101 off iGEM10_042 and 041 in hybrid vectorAnalytical Mapping: Lane 1: iGEM10_042, expected length: 2863bp The PCRs look successful. I'm going to Eco/Bam the PCR product and ligate with pMLL4AC. Cotransformation Results for iGEM10_019 and ef1a/atub+2093iGEM10_019: only sample c was co-tranformed, but I'm only going to miniprep b and d, since I forgot to streak sample a on the antibiotics plate. All of the samples for ef1a/atub+2093 were co-transformed. I'll pick a lot more colonies today, colPCR, inoculate, and streak for cotransformation. Amy N. Kristofferson 14:37, 22 July 2010 (EDT)To do:
Nuclear Localization checkpjc012 backbone Bgl/XhoI transfer of CDS of our protein his6tag prom-rbs-his6-my gene! rfp+nls: 1884+1858 in 1601KC Transformed into jtk049. Started rescue at 3:35pm. Plated on KC. Tomorrow, pick colonies. Can do colPCR off plasmid (1601 series). Next day: miniprep, map, sequence? PCR on bgl/xhoI sites. may need to pcr a new bam/xhoI around GFP-Nls and then cut into mike's plasmid Sequencing Results for iGEM10_047, 48, 51, 52 (SL/VB Assembly Product with L-Lysozyme Self LysisAll parts look perfect for about the first and last 1000bp.
Picking ef1a/atub-Bjh2093 in pMLL/1601KC hybrid vector colonies
Picking iGEM10_019 coloniesThe plate looks great. It's covered in fairly healthy looking colonies.
If both 17 and 16 (a part of 18) are there, the colPCR will produce a band ~888bp long.
ColPCR results from above colony picksLane 1-4: iGEM10_019 w/ igemTen21/24, I wanted 888bp band, so they all look great! Even though my ef1a/atub+2-93 colPCR results don't look so hot, I'll miniprep the ones that weren't co-transformed and map them tomorrow. ColPCR results for ig114/2271+2017 picksThis analytical backbone PCR seems useless, since a 900bp band was supposed to reveal the presence of the p15 origin of rep, a sign that the part was in the hybrid vector but my negative control, a part in pMLL4AC, also produced a 900bp band! My only choice of analysis then is mapping, but all of the colony PCRs look very crazy. Maybe I'll just miniprep and map them all anyway. Another route I could do would be to perform an Eco/Bam transfer into a vector w/ diff antibiotics, and then do another Eco/Bam transfer into the PMLL4AC. That's just going take a long time... th395/th396
ca998/g00101
One of these is clearly still in its hybrid vector, but I don't know what's going on with the single bands. I'm going to Fusion PCR off ___ using ca998 and g00101. Then I'll Eco/Bam that into PMLL4AC. Amy N. Kristofferson 13:04, 21 July 2010 (EDT)ig114/2271+2017 vector transfer redoPick 4 colonies for ig114/2271+2017. Normal colPCR seems to be a waste of time, but I'll try it anyway. I'm also going to do my backbone analysis PCR. Negative control: Bjh2345 in PMLLAC.
Assembly of iGEM10_018 and 017 to make iGEM10_019Manual R/L Digest of iGEM10_018 and 017. Started at 10:00am. Zymo'd. Ligation started at 1:08pm. Transform into Righty cells. Rescue started at 1:50pm Plated on KA. Labelled plate iGEM10_019. One Pot Assembly of atub/ef1a with jh2093DNA used: Rigthy: Bjh2093 in 1601AC Lefty: ef1a PMLLKA b (7/14 miniprep) and atub PMLLKA b (7/14 miniprep) All samples are methylated. NOTE: I'm intentionally creating a hybrid vector! I think this will save time. Just make sure not to transform into Righty, because then I won't be able to Eco/Bam the product into a good vector. Also, this ef1a has a two base pair deletion, so it may not be functional. Digest started at 1:32pm. Heat kill at 2:49pm. Add ligase at 3:15pm. Add jtk030 cells and heat shock at 3:45pm. Start rescue. Plate at 4:49pm.
1uL lefty plasmid(methylated) 1uL righty plasmid (methylated) 1uL NEB2+ATP 0.3 uL XhoI 0.3 uL BglII 0.3 uL BamHI 6.1uL Water
Amy N. Kristofferson 13:45, 20 July 2010 (EDT)None of the colonies picked yesterday from the iGEM10_047, 48, 51, 52, or 16+2294 were co-transformed. And since the ColPCR didn't reveal much of anything, I'm just going to miniprep the first two samples of each, except for iGEM10_047. iGEM10_047's d sample looked great on ColPCR, so I'll miniprep sample a and d instead of a and b. Also, somehow all 12 of the colonies that I picked for the transfer of 2271/ig114+ 2017 from the hybrid into PMLL4AC seemed to still be in the hybrid vector, based on my analytical pcr (they all had bands around 900bp). I'll miniprep four of each anyway and map them. If they're not the right side, I'll do the transfer over again. To do:
Picking colonies w/ Payload and Payload delivery30µL into ea mL TB media (use TB for growing up for assay) Miniprep Map
Submitted a sample a for each of the 2345/1968 assemblies for sequencing, except I submitted sample d for iGEM10_047. 16+2294 is righty methylated. That's why I only got one band. It's the right sized band though. I'll map it with Eco/XhoI. Started digest at 4:10pm. Lane 1: 16+SLa Expected lengths: 4644, 1466 Analysis: I think the top band is parent vector. Don't know why sample B has three bands... very strange. I submitted a for sequencing. Redo of Hybrid vector transferThose oligos shouldn't PCR off the genome (http://archaea.ucsc.edu/cgi-bin/hgPcr?hgsid=462798&org=Escherichia+coli+K12&db=eschColi_K12&wp_target=genome&wp_f=cggttatccacagaatcaggg+&wp_r=gcgctgatgtccggcggtgc&Submit=submit&wp_size=8000&wp_perfect=15&wp_good=15&boolshad.wp_flipReverse=0) I'm going to make more of pMLL4AC Eco/Bam digest from Tim's stock in plate 2, well F7 (pMLL4AC Bth8062). Started digestion at 2:04pm. Drop out is 813bp. Vector should be 2745bp. Use 3uL of digested pMLL4AC instead of 1uL when ligating. Unfortunately, there's only about 1.75uL of digest PMLL4AC vector left, so I used it to redo the ligation w/ eco/bam digested ig114+2017. I started the ligation at 1:51pm. Started rescue at 3:04pm. Redo of ligation with new pre-digested and gel purified PMLL4-AC w/ FFGFP dropout. Started ligation at 4:45pm. Started rescue at 5:30pm. Amy N. Kristofferson 14:51, 19 July 2010 (EDT)Everything grew up very well. The only concern I have is that the colonies with 2345 in the part have a transparent loop around the colony, possibly suggesting that self-lysis is starting. I'll have to careful when I miniprep these. I pick colonies, ran a normal colony pcr for all of them, ran an analytical pcr to make sure the parts I transfered out of the hybrid vector aren't still in the hybrid vector, inoculated and check for co-transformation for the assembly plates. Here are my notes:
Analytical PCR results for Transfer from hybrid vectorThis is a gel from a colPCR with oligos th395/th396 of four colonies picked from 2271+2017 and ig114 7/16/10 plates. If the plasmid is still a hybrid, it will contain the p15 origin of replication, and will PCR off a ~900bp DNA. This gel suggests that all the colonies I picked contained the hybrid vector. Tim said that if I need to do the transfer over again, I should use more pMLL vector (~3uL). For now, I'll pick more colonies.
ColPCRThis results aren't very good. But I think that's to be expected when parts are about 6k or greater. I'm going to mostly ignore these results and miniprep a couple samples from each reaction anyway. The analytical PCR makes it seem like all the hybrid transfers are still in the hybrid vector... :(
Amy N. Kristofferson 15:20, 16 July 2010 (EDT)To do: -Eco/Bam digest ig114+2017 g.p. and 2271+2017 g.p. Run on gel and gel purify. Ligate to pMLLAC pre-digested vector. (calculated expected lengths before running gel, to make sure it's worth the trouble. Bands might be so similar that maybe just a zymo is better) -ligate g.p. 2294 with g.p. 16a. -assemble SL-L with vacuole buster:
NOTE: can't use one pot because iGEM10_043 and iGEM10_044 are NOT methylated. I transformed jtk030 cells to make them. I'll have to do separate righty and lefty digests. Sequence Analysis of Ef1a/Atub
So where to go from here? I'll submit atub for sequencing with g00101. As for ef1a, since my ef1a in the AK vector (was supposed to be KA... argh) sequenced perfectly. I'll just Eco/Bam Transfer it? Then I'll get colonies tomorrow, and I can ColPCR/inoculate and then miniprep on Tuesday. OR I could pick more colonies today. However, even if I map well, the part could have mutations... Transfer of 2271/ig114+2017 to PMLLAC from pMLL/1601 hybridEco/Bam digest of 2271+2017 g.p. a, ig114+2017 g.p. a. Gel purify part: 2271+2017 in pMLL/1601 vector Eco/Bam: 3670, 2704 Lower band includes parts. ig114+2017 in pMLL/1601 vector Eco/Bam: 3670, 2934 Lower band includes parts When I pick colonies, I'll run two ColPCRs: ca998/g00101 and th395/th396. If the plasmid is still a hybrid, it will contain the p15 origin of replication, and will PCR off a ~900bp DNA. I'll also run the ColPCR on colonies that I know contain the hybrid so that I can compare. This will allow me to do less minipreps. I'll still map them though, to confirm the vector size.
TH395 gcataGAATTCcggttatccacagaatcaggg fwd for EcoBAM p15A, pBth7011 TH396 ctaggGGATCCgcaccgccggacatcagcgc rev for EcoBam p15A, pBth7011 Second Step of Assembly for SL/VB: L-1968/2345 to iGEM10_043/044Bam/XhoI: 2345 in PMLL4 b, 1968 in PMLL4AC (T1, L) Bgl/XhoI: 2114+2316 g.p. a, 2114+2275 a (7/14) Transposase Assay: ligating gel purified 16a to gel purified 2294 in CORRECT KA vectorDetails of today's Digestions, Ligations, Transformations, and Platings
Submit 2114+2275a 7/13 miniprep for sequencingSubmissions:
Results: Great! This part is as it should be. Sequencing Results for ig114+2017
I'm going to Eco/Bam Transfer the part, because it's currently in a pMLL-jh1601 fusion backbone. Amy N. Kristofferson 14:55, 15 July 2010 (EDT)To do:
Important Mini-meeting update: Issue with SL Assembly probably resulted from use of 2017 in 1601 vectorMapping Yesterday's MiniprepsRan out the gel a bit more, and I think it shows that the two 2271+2017 samples mapped well:
Sequencing Results for the rest of the Minipreps from my first batch of colony picks from Gel-Purified Assembly
The boundaries of parts 2114+2275 and ig114+2017 look good. I'm going to PCR off the middle to check if the insides are go too. I used ca56 for ig114+2017 (binds to pbad promoter). I designed a primer for the middle of Amy N. Kristofferson 13:24, 14 July 2010 (EDT)To do:
Sequencing Submissions: the rest of my gel-purified assembly miniprepsiGEM51-56= each part w/ ca998 and g00101 Cotransformation RedoRescue started at 11;32am. Sequencing Results for 2114+2316 (iGEM10_043): Great news! Successfully put Pcon in front of VBaI submitted the part with ca998 and g00101, and the results clearly show that the Pcon is in front of the vacuole buster. Woo hoo! KA transferChristoph Miniprepped everything he needed. I miniprepped the remaining 1mL of 10 or bjh2294-PMLLKA (eluted with 25uL to keep normal MP concentration), ef1a-PMLLKA b, atub-PMLLKA b,c. Parent Vector bleed-through results: All samples from 1,2,3,4,6,7,8,12,13,16 are good. The only samples with co-transformation were 15a and 9a,b,d.
Colony PCR- ca998+g00101
NOTE: lanes 49-96 refer to the middle gel. Just subtract 48 from the lanes in the table to see what number to refer to on the actual gel. Colony PCR-th313+iGEM10Note: the results for 5 are in the middle right gel shown above.
SL/VB Assembly Colony PicksCotransformation results: 2h and 6a were co-transformed. Everything else had no growth on the triple antibiotics plate.
Amy N. Kristofferson 18:16, 13 July 2010 (EDT)Co-transform jh2348/2291-1601K with Tera's PayloadAdded 100uL of H2O to 1uL of DNA from Tera's Ef1a and Atub payload constructs. Added 1uL of each of those dilutions to 70uL of MG1655 cells. Mg needs to be added for all steps of cloning the PDD's 1M MgSO4 10µL into rescue step (after heatshock) 400µL spread on plate (if +Spec, add 20µL and spread) 30µL into ea mL TB media (use TB for growing up for assay) Started rescue at 4:06pm. KA Vector transfer: ColPCR (parts and vector backbone check), inoculate, grow upI picked colonies, inoculated and ran a colony pcr for all the parts we moved into the new, CORRECT KA vector. Important notes:
SL/VB Assembly: Picking colonies from 7/6, 7/7, and 7/8 platesPicked 8 colonies from Assembly attempt #2 (one pot) and assembly attempt #3 (gel purification) for 2114+2275, ig114+2017 and 2271+2017. Inoculated, checked for co-transformation, and set up ColPCR. Amy N. Kristofferson 14:43, 12 July 2010 (EDT)IMPORTANT UPDATE: TUBE MARKED PMLL6 (KA) in Eco/Bam Box is INCORRECTChristoph just ran and analytical digest/gel that proved that the vector in the tube that was supposed to contain KA actually contained AK vector instead of KA. I need to Eco/Bam transfer the parts into a new KA vector. Also, this explains why the assembly with the Self-Lysis Device didn't work. We need to Eco/Bam transfer it into a KA vector too. Digesting the new pMLL-KA backboneMarianna gave us 10uL of her CMED7 in KA part. The dropout will be 324bp. To digest all of it, I multiplied the Analytical Mapping protocol by 2.5: 10uL of Water 10uL of CMED7-PMLLKA Miniprep 2.5uL NEB 1.25uL EcoRI 1.25uL of BamHI Total: 25uL Load 12.5uL into 2 wells. Gel Purify. Elute each with 12.5uL of H20. Started digest at 4:30pm. Ligation of ef1a and atub (bjh2342) eco/bam zymo'd digestLigated ef1a and atub eco/bam zymo'd digests (from my previous insertion into the supposed KA vector) with the new gel purified Eco/Bam digested KA vector. Started at 7:05pm. Transformed jtk030, MC1061 pir+ cells. In retrospect, I could have used Lefty. Checking vector backbones of SL/VB Eco/Bam transfersSet up a 2K55 PCR reaction with Taq MM, 1uL of 1/10 th313 oligo (in the middle of AmpR) and iGEM10 (rev oligo right before EcoR1 site) to verify the order on antibiotics on the backbone. This reaction PCR's off the AmpR gene to right before the EcoRI site. So if the vector is actually A-, the PCR product will contain a bit of AmpR and plasmid backbone and will be 865bp. If the plasmid is -A, the PCR product will contain a bit of AmpR, lots of plasmid backbone and the other antibiotic. In the case of CA, this amounts to 1808bp. See Eco/Bam page in Excel notebook. This data suggest that all the backbones that should be A- are indeed A- and that all the backbones that are -A are indeed -A. So the antibiotics shouldn't be the problem. For whatever reason, nothing turned up in the 2345 lane. I'll run the PCR again just to double check.
Lane Part Color Expected length Good? 1 Bjh2345a AC 865 no-empty lane 2 Bjh2345b AC 865 good 3 Bjh2345c AC 865 good
Analytical digests of SL/VB Gel Purf. Assembly Minipreps
Payload Delivery: Analytical Mapping of 2348 and 2291 in 1601KBoth parts look great! I'm going to give them to Jin.
Eco/Bam Transfer from Bad KA to Good KA backbone
Amy N. Kristofferson 21:13, 10 July 2010 (EDT)Must do today:
If I have downtime, could start items in the above To Do list From yesterday's colony picks, only the first colony picked from 16a H3 was cotransformed. No idea how that happened, since I gel purified the digests before ligating. Amy N. Kristofferson 13:37, 9 July 2010 (EDT)Colony PCR of SL/VB and 16a+SL AssemblyResults: Very odd. Even though I gel purified the correct bands, it appears that only 2114+2316 worked correctly. I'm going to check to make sure that everything is in the correct vector, because I don't know what else could be causing a problem. Since I'm miniprepping a the couple correct products, might as well miniprep and map a few others to try and figure out what the problem may be.
Animal Promoter: Analytical Mapping of atub and ef1a promoter in pMLLKAStarted an Eco/Bam digest of the 4 minipreps I made of each promoter (all of which had GREAT colony PCRs) at 10:35am. They will be ready to run on a gel at 11:05am. Once it has been verified that these parts map well, I'll submit them for sequencing and then begin the next step of assembly.
Submitted ef1a-a and atub-b Sequencing submission:
Amy N. Kristofferson 14:36, 8 July 2010 (EDT)Cotransformation checkNone of the 2271+2017 or ig114+2017 colonies were cotransformed, but ALL of the iGEM10_018 (16a+SL) were cotransformed. Colony PCR results for 2271+2017, ig114+2017 atub and ef1a promoter, iGEM10_16aCan disregard the results for 16a+SL since all samples were cotransformed. 2017 combo inoculations are useless since nothing showed up on the colony pcr. Very strange, since they weren't co-transformed. GOOD NEWS: atub and ef1a look great.
Redo of SL/VB and 16a+SL digests with Gel PurificationTo avoid co-tranformation and hopefully encourage the creation of the parts I want, I'm going to run a preparative gel, select the appropriate bands and gel purify. Performed a Lefty (Bam/XhoI digest), Righty (Bgl/XhoI digest), and one-pot digest on all the parts in my SL/VB promoter as well as iGEM10_016a and Bjh2294 in PMLL-KA can be found in the Cheshire Chat plate in H3 and F3. I'll gel purify the Lefty digests of Bjh2345, Bjh2271, Bjh1968, Bjh2114, ig114, 16a and the Righty digests of Bjh2316, Bjh2275, and the two self-lysis devices. The other digests are just to make sure everything was methylated correctly and that the one-pot reaction works on these vectors. Notes on protocols: Used Manual 2ab Assembly protocols for Lefty and Righty Mastermixes. Used my One-pot recipe, except with 0.4uL of each enzyme. Also, the DNA taken from the Cheshire Cat Plate was diluted by half b/c it was eluted with 100uL, so we had to adjust the amounts of NEB2 and enzyme used so that the recipe in the R/L digests turned out to be 1.6uL NEB2 and 1.5uL of each enzyme total, since our using 8uL of DNA instead of 4uL of DNA would otherwise have diluted the buffer and enzyme. Digests started at 3:35pm. From left to right: Lefty Digests, Righty Digests Triple Digests Results:
NOTE: lane 2 in the lefty gel is Bjh2294 H3, and everything else is pushed over one lane Summary: Everything is methylated as it should be. I cut out all the right bands and purified them. Also, this shows that triple digestion isn't very efficient and cause a lot of parent vector bleedthrough (the top band of all the lanes). Also, note how dilute the SL DNA is... I'll still elute with the standard 8.5uL to concentrate the DNA, but this is still a potential problem. Ligation and Transformation of SL/VB and 16a+SL purified digestsStarted ligation at 6:45pm. Will transform into jtk030 (pir +, MC1061 cells). I forgot to digest 2017, so I used a righty digest that I had done previously. Started rescue at 8:10pm. Amy N. Kristofferson 13:45, 7 July 2010 (EDT)All the samples from 16a,b + SL were cotransformed, and since Jin mentioned that making a mastermix for the onepot reaction may result in there not being enough enzyme per reaction, I'm going to do it over again without making a mastermix AND using 0.5uL of enzyme instead of o.3uL of enzyme. This should cut back on parent vector bleedthrough as well.
SL/VB Assembly: Redo of One Pot (with more enzyme) for 2114 combos
iGEM10_020 Assembly: redo of 16a+SL assembly, starting with new L/R digests
Possibly successful assemblies (Animal Promoter, SL/VB and iGEM20
Miniprep of Payload Delivery VectorsMiniprepped Payload Delivery stuff (in shaker w/ Mg LB)
Sequencing Results for iGEM10_017b, 2114+2316b,Bjh2345 PMLL4Sequencing results:
Amy N. Kristofferson 13:49, 6 July 2010 (EDT)Colony PCR results for all colonies picked todayNote: they ALL look horrible. Bands in the 16a lanes are probably from cotransformation with 16a vector, since that part is about 850bp.
Transposase Assembly: Colony PCR of iGEM10_016a,b with Self Lysis DevicePicked 4 colonies of each. Ran Colony PCR. Inoculated. Plated to check for co-transformation. Animal Promoter: Colony PCR of Ef1aPicked 2 colonies. Ran Colony PCR. Inoculated. Conor Eco/Bam digested and zymo'd the atub promoter (Bjh2842). Started ligation with PMLL6-KA vector at 4:45pm. Transformed into jtk049 cells. Started rescue at 5:55pm. Payload DeliverySince the payload delivery device contains the Self Lysis device under a promoter that's induced in low levels of Mg, I need to grow up my colonies in LB containing 30mM Mg to prevent Lysis. In the future, I should also rescue/plate on Mg. I selected clear colonies. Tomorrow, I will miniprep the inoculations and map the minipreps next to the parent vector containing the part, so I can make sure the whole part is in the new vector. (Jin said recombination events can occur which could shorten the part) SL/VB Promoter AssemblyRedo one pot reaction with 2271+2071 and ig114+2071Conor set up two one pot reactions and put them in the thermocycler under my ONEPOT program. Added ligase at 4:45pm. Will transform into LEFTY cells. Started rescue at 5:55pm. Picked more colonies for 2114+2275, since miniprep mapping failedPick 8 more colonies from 2114+2275. Ran colony PCR, inoculate, check for cotransformation. Summary of Results so far
For whatever reason, my Colony PCR results and miniprep results don't seem to ever correspond. Amy N. Kristofferson 19:16, 5 July 2010 (EDT)SL/VB Promoter Assembly: Mapping the minipreps produced from Round 1 and 2345 RedoStarted digestion at 5:18pm. Will be ready for the gel at 5:48pm.
Transposase Assembly: Ligation of iGEM10_016a,b Lefty digest with 6+24 Righty digestStarted ligation at 4:05pm. Ligated unmethylated 6+24 digested with BglII+XhoI with both unmethylated iGEM10_016a+b, which had been digested with BamHI+XhoI. Tubes are labelled 16a+SL and 16b+SL (SL meaning Self Lysis, which is part 6). After ligation is over, I will transform into RIGHTY cells. Started rescue at 5:05pm. Plated on CA. Animal Promoter: Ligate Ef1a Eco/Bam digest of PCR product with PMLL6-KAStarted ligation at 4:05pm. Ligated Ef1a Eco/Bam digest of PCR product with pre-digested PMLL6-KA. After ligation is over, I will transform into LEFTY cells. Started rescue at 5:05pm. Plated on KA. Payload: Ligation of Bjh2348 and 2291 with Pjh1601KStarted ligation at 4:05pm. Ligated Eco/Bam digests of Bjh2291 and 2348 with my Eco/Bam digested Pjh1601K plasmid. After ligation is over, I will transform into jtk049 cells, because they don't need to be methylated. Started rescue at 5:05pm. Plated on K. Amy N. Kristofferson 13:00, 2 July 2010 (EDT)Transposase Assay Take 2: Testing time for Transposase expression and time for transposase activity
Unfortunate update: Sequencing proved that iGEM10_020b is a dud. Gibson didn't work because one of our junctions contained palidromic terminal repeats, so the oligos designed based off those junctions didn't have a unique binding site. Oh well. We'll proceed with manual 2ab assembly. iGEM10_20 2ab AssemblyMapping of iGEM10_016 minipreps a and bAn Eco/Bam digest of iGEM10_16 in PMLL5-CK should result in bands at 2662 and 867. Note: iGEM10_017 has already been mapping. (see below) Results were slightly confusing, so I'll submit it for sequencing...? iGEM10_001 was also mapped, and looks good. 1a-c are all good options. Started digestion at 4:55pm. Both have bands at the appropriate places, but 16b has a lot more parent vector remaining, for whatever reason, so 16a is probably the best choice. Assembly of iGEM10_018Lefty Digest of iGEM10_16 Righty Digest of Self Lysis Device (6+24 taken from well G10 of ROMEO plate) Started at 4:42pm. Zymo'd and stored in my working box. SL/VB Promoter Manual Assembly: Analysis of One-Pot reaction2114+2316 and 2114+2275 both successfully transformed righty pir+ cells. The plates are covered in colonies. 2271+2017 and ig114+2071 weren't as successful. The former had no colonies, while the latter had 11, which is surprising, because I used barely any DNA. I will pick four colonies from each, colony pcr, inoculate, and check for cotransformation. Cotransformation results: The ones with letters in the corners were chosen for minipreps.
Moving into jh1601KPerformed an Eco/Bam Digest of jh1601K. Put in incubator at 11:09am. Trashed that digest. Realized I need to do a gel purification, so I'll do the digest again with 4uL of plasmid. Started digest again at 4:54pm. 2345 Redo from 24+PMLL4 digestsThe plate is covered in colonies. I will pick 8 colonies, inoculate, and check for cotransformation.
Amy N. Kristofferson 13:40, 1 July 2010 (EDT)Eco/Bam transfer 2348 and 2291 into 1601KStarted Digest at 12:19pm. Zymo'd and stored in my working box.
2345 Redo: Ligating Tim's ebid 24 and EB pMLL4-ACSince my Colony PCR turned out so horribly yesterday, and 2345 is the Self Lysis Device we've been using for all our construction, I'm just going to use Tim's digests and ligate them together. Started ligation at 11:48am. I transformed the plasmid into lefty pir+ cells. Started rescue around 12:30pm. Plated on AC at 2:13pm. One Pot Assembly of Jin's PartsI'm going to redo the construction of the following with a one pot reaction, since they're all methylated.
Started digest at 11:10am, for all but ig114+2017. I added the speck of 2017 (~.2uL) left at around 11:36am. Started the 20 min heat kill for all but ig114+2017 at 12:30pm. Accidently set the heat kill program for 20 sec instead of minutes and added ligase. Hopefully that won't hurt anything. Put all four tubes in the PCR machine to heat kill at 12:53pm. Added ligase at 1:41pm. Started rescue at 2:42pm.
These numbers have been based on Qiagen minipreps of Jin's pBjh1601
assembly vectors:
1uL lefty plasmid(methylated) 1uL righty plasmid (methylated) 1uL NEB2+ATP 0.3 uL XhoI 0.3 uL BglII 0.3 uL BamHI 6.1uL Water
Notes:
tubes labeled with a red slash
distribute the plasmids into it
1hr at 37 and heat kill steps of the procedure Eco/Bam digest of EF1a PCR productStarted digest at 11:55am. Zymo'd the product. I need to figure out what assembly vector to put it in and then map and sequence the part to make sure it's correct. |