Berk2006-LocksAndKeysTeam

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KAD_07/12/06

• sequencing confirmed accuracy of pJ23006, pSB1A2-J01129, pJ23006-I13522-1, and pJ23006-I13522-2, but showed that pSB1A2-J01122-1 was the wrong clone.
• miniprepped and sent pSB1A2-J01055 and pSb1A2-J01020 (from Box2) for sequencing
• transformed 994library into TG1 cells
• grew liquid culture of pSB3C6-J01122-A3 (iG021) in order to make competent cells
• screened more Key3 colonies
• made pSB3C6-j01122 competent cells.
• cotransformed j01122 x j01129
• resubcloned J23007, 8, 9 and plated
• threw away old pJ23006, pJ23006-I13522
• transformed 994library into 1122

KAD_07/11/06

• digested pJ23006-J23009-1 and pJ23006-J23009-2 with BamHI and AlwnI for analytic gel mapping. results from this gel show both versions of pJ23006-J23009 were in fact parent vector. these results imply that the long fragments from yesterday's gel are the correct clones, contrary to what we had first thought. we will need to colony pcr at least 4 more colonies of pJ23006-J23009 in order to find the correct clone.
• -80 stocks of pJ23006-J23007-3 and pJ23006-J23008-2 have been made, and both clones were also sent for sequencing.
• still waiting for seqencing results from 7/7 and 7/10, so we have yet to confirm accuracy of Lock 1 (J01063), Lock 2 (J01068), pSB3C6-J01122-A3, pSB3C6-J01122-A4, and pJ23006, as well as pJ23006, pSB3C6-J01122-1, pSB1A2-J01129, pJ23006-I13522-1, and pJ23006-I13522-2.
• scraped plate and mini-prepped pJ23006-994library
• sequencing confirmed the accuracy of pSB3C6-J01122-A3 and pSB3C6-J01122-A4, so the A3 clone was transformed into TG1 cells.
• grew cultures of key 1 and 2 that were plated yesterday: J01055 (on amp) and J01020 (from box2 on amp)

BCH_07/11/06

colony pCR to screen Keys, again
Oligos used: ca998 and G00101 pJ23006-J23007: 487 if clone, 1381 if parent vector
pJ23006-J23008: 338 if clone, 1381 if parent vector
pJ23006-J23009: 556 if clone, 1382 if parent vector
gel map1: latter, 7.5, 7.6, 7.7, 7.8, 7.9, 7.10, 7.11, 7.12
gel map2: latter, 8.5, 8.6, 8.7, 8.8, 8.9, 8.10, 8.11, 8.12
gel map3: latter, 9.5, 9.6, 9.7, 9.8, 9.9, 9.10, 9.11, 9.12

to be done on tuesday:

• scrape 994 library plate and miniprep it.
• if J01122 sequence data has been received then make competent cells of these.
• all sequence data received needs to be filled in the wiki and noted on the miniprep tubes.
• run an analytical gel on pJ23006-J23007-2, pJ23006-J23007-3, pJ23006-J23008-1, pJ23006-J23008-2, pJ23006-J23009-1, pJ23006-J23009-2 minipreps to determine the correct clones. Digest with BamHI and AlwnI but double check this in Ape. sequence the ones that come out correctly.
• pick colony from the pSB3C6-J01122 plate and grow a liquid culture to miniprep and -80 stock it assuming sequence data comes back groovy.
• transform 994Library, J23007, J23008, and J23009 into the J01122 competent cells that were made.
  

BCH_07/10/06

• Subcloned 994 library into pJ2306-I13522 to make pJ23006-994Library. They are plated on a big plate in incubator.
• Screened pJ23007, 8, 9 by colony PCR and results need to be put up by kaitlin.
• pJ23006-J23007-2, pJ23006-J23007-3, pJ23006-J23008-1, pJ23006-J23008-2, pJ23006-J23009-1, pJ23006-J23009-2 were minipreped and need to be sequenced.

KAD_07/10/06

• miniprepped pJ23006-I13522 verions 1 and 2
• plated pSB3C6-J01122-1 from liquid culture
• sent pJ23006, pSB3C6-J01122-1, pSB1A2-J01129, pJ23006-I13522-1, and pJ23006-I13522-2 for sequencing
• still waiting for sequencing results of Lock 1 (J01093), Lock 1 (J01068), pSB3C6-J01122-A3, pSB3C6-J01122-A4, and pJ23006
• plated J01055 on AMP from -80 stock
• plated J01129 on AMP from -80 stock
• plated versions of J01020 from -8o Box 1 and Box 2 on AMP, KAN, and CAM plates, because no information could be found relating to the part's antibiotic resistance

colony pCR to screen Keys
pJ23006-J23007: 487 if clone, 1381 if parent vector
pJ23006-J23008: 338 if clone, 1381 if parent vector
pJ23006-J23009: 556 if clone, 1382 if parent vector
gel map: latter, 7-1, 7-2, 7-3, 7-4, 8-1, 8-2, 8-3, 8-4, 9-1, 9-2, 9-3, 9-4
Of all versions of pJ23006-J23007, only clone 3 appears to have cloned correctly. The bands only differ slightly, however, so we will miniprep a band of each length just to be sure. (7-2, 7-3)
Of pJ23006-J23008, we had similar results, so we will miniprep a versions of each (8-1, 8-2).
Of pJ23006-J23009, all bands were the same short length. We will miniprep 2 to be sure that these results were accurate, otherwise more colonies will need to be screened in order to find the long variety. (9-1, 9-2)
7-3 and 8-2 were large variants, all others were short bands.

• there are two different clone versions for pJ23006-I13522. For the construction of pJ23006-994Library, i am using pJ23006-I13522-1. note if the sequence is incorrect for this clone, the construction must be redone. Also, make sure to throw out -80 stock of pJ23006-I13522 if sequence data comes back incorrect. Throw away pJ23006-I13522-2 if pJ23006-I13522-1 comes back correct. -BCH

KAD_07/07/06

• miniprepped cultures of pSB3C6-J01122 (versions A1, A2, A3, and A4)
• miniprepped Lock 1 (J01063) and Lock 2 (J01068)
• analytically digested and mapped pSB3C60-J01122-A1, pSB3C6-J01122-A2, pSB3C6-J01122-A3, and pSB3C6-J01122-A4. results on the gel suggest that all four versions were correct, and most likely have the right genetic sequence.
• then sent Lock 1 (J01063), Lock 2 (J01068), pSB3C6-J01122-A3, pSB3C6-J01122-A4, and pJ23006 out for sequencing

BCH_07/07/06

• colony PCR to confirm subclone of pSB3C6-J01122:
  • using oligos ca1000 (forward, cagggcagggtcgttaaatagc) ca1001 (reverse, ggcggttttttcgttttcagagc). These are sequencing oligos for pSB3C6 (pAC997).
  • Parent vector will product fragments of size 173 bp, whereas the cloned vector will produce fragments of 1101 bp.

• Colony PCRs ALL came out perfect. I grew up clones 1 and 2 and minipreped them both. we will sequence 1 and keep 2 until it's confirmed.
• pJ23006 is growing up to make -80 stocks. this needs to be minipreped and potentially sequenced again because we ran out.
• Subcloned key3b, key3c, and key3d (J23007, 8, 9) into pJ23006.
• Subcloned I13522 into pJ23006. 994 library needs to be subcloned and transformed after this is screened and minipreped.

KAD_07/06/06

• sequencing data confirms accuracy of pJ23006, but also shows that both pSB3C6-J01122 and pJ23004 do not match expected results.
• disposed of -80 stocks of pSB3C6-J01122-1, pSB3C6-J01122-4, pJ23004-1, pJ23004-4, pJ23006-1, and pJ23006-2.
• grew up 4 new cultures from original plate of pSB3C6-J01122 (grown 6/30/06)
• grew cultures of Lock1 and Lock2 from plates grown yesterday
• minipreped pJ23006, however this still needs to be sequenced.
• subcloned and transformed pSB3C6-J01122 yet again and im not sure if we need to sequence this. needs to be verified some how (colony PCR)

KAD_07/05/06

• still waiting for sequencing confirmation of pSB3C6-J01122, pJ23004, and pJ23006.
• digested 994 key3 library, J23007 (key3B), J23008 (key3C), J23009 (key3D), and pSB1A2-I13522 (to make J23006-I13522 with GFP instead of RFP)
• made overnight cultures of pSB3C6-J01122-1, pSB3C6-J01122-4, pJ23004-1, pJ23004-4, pJ23006-1 and pJ23006-2 in case original minipreps cannot be located.
• gel purified pSB1A2-I13522 digest, small fragment clean-up of J23007, J23008, J23009, and 994 key3 library to prep for ligation and transformation to test key3 variants.
• plated Lock 1, Lock 2, pSB3C6-J01122-1, and pSB3C6-J01122-4.

DLK_07/03/06
-80 Stocks of pSB3C6-J0112 #1, pSB3C6-J0112 #4, pJ23004 #1, pJ23004 4, pJ23006 #1, pJ23006 #2 created and stored in box3.

KAD_07/03/06
Today we miniprepped overnight cultures of pSB3C6-J01122, pJ23004, and pJ23006.
We then mapped each plasmid:

• cut pSB3C6-J01122 with EcoRI and PstI to produce fragments 2042 and 974 bp. (Parent vector would yield one 3203 bp fragment)
• cut pJ23004 with SpeI and PstI to produce fragments 2135 and 955 bp. (Parent vector would yield two fragments: 3066 and 14 bp)
• cut pJ23006 with EcoRI and XbaI to produce fragments 3065 and 214 bp. (Parent vector would yield two fragments: 3065 and 15 bp)

Results from the analytical digest suggest that clones have correct respective sequences, but nothing can be confimed until sequencing results are received and analyzed.
Lanes 1-4: pSB3C6-J01122, Lanes 5-8: pJ23004, Lane 9: ladder
Lanes 1-4: pJ23006, Lane 5: ladder

BCH_06/29/06

• Sequence data for the -80C stock shows that pSB3C6-J01122 is in fact the parent vector pSB3C6. We suspect this is sue to a tube switch early on in the project.
• We extended the keys that we fabricated (parts b, c and d). they are in the riboregulator -20 box and have been purified from their extension reactions.
• We ran an assay on the 1122 and 1129 constructs on the tecan and found bad results. the key+lock transformation showed absolutely zero increase in flouresence suggesting that the key doesnt unlock shit. We later found out that the lock, 1122, that we thought o be working perfectly was in fact the parent vector, pSB3C6. Some tube switched went down and we had to throw out pSB3C6-J01122 mini and -80 stock, all -20 tubes labeled pSB3C6-J01122 or pSB3C6-J01022, or J23000 or j01129X. it was a massacre.
• Kaitlin cut out J01122 from pSB1A2-J01122 and ligated it into pAC997 (pSB3C6) to make pSB3C6-J01122.
• Trashed pSB3C6-J01122 competent cells (-80 stock).
• Kaitlin resubcloned and transformed pSB1A2dXS into TG1s, and they are on a plate and incubating.
• Subcloned and chris transformed J23006 (Ptet-X-GFP-S).

KAD_06/29/06
Tecan scan results:

• Lane 1A: CAM, J01022-B
• Lane 2A: LB + CAM, J01122-A
• Lane 3A: Carb + Kam, J01129 + J01122
• Lane 4A: LB, TG1 Cells

A	1	2	3	4
A	13357	104	71	71

WRB_06/28/06

• colony pcr 994/TGI's; colony pcr -> analytical gel to check key3 library to verify presence of key. The gel results showed the expected size in 9 out of 10 cases. The 10th case was not even close.
• transformed psb1A2_dXS into TG1 cells (chemically competent); the last attempt was unsuccesful.
• started overnights of psB3C6-J01122, -J01124, -J01022, and psB1A2-J01129

DLK_06/26/06

• Evening ligation and transformation
  • The purified DNA product from Kaitlin was ligated using the standard ligation procedure. Additional 5.5ul backbone (6.5ul total) was subsituted for 5.5ul water

• pSB1A2dXS, pGLN1003, DNA-2/23H,DNA-2/23F were transformed into TGI, with KCM and plated onto Amp plates. A slightly modified transformation procedure was used:
  • A 280ul cocktail was created
    • 200 ul TGI
    • 30 ul KCM
    • 50 ul h20
  • 1ul of the plasmids (DNA-2/23f DNA-2/23h) was added to tubes (single plasmid per tube, 40ul cocktail) and 10ul of ligation (pSB1A2dXS, pGLN1003) was added to tubes. (single ligation product per tube 100ul cocktail) 4 tubes total, each with single vector.
  • 10min on ice, 42C heat shock for 1.5min, 5min on ice, plated

BCH_06/26/06

• Minipreped and sequenced pSB3C6 J01122-A
  • Sequence data from what we thought was J01122 came in and it turned out that it was in fact unlocked RFP (J01022) thereby resulting in constitutive RFP production. As such, we minipreped the actual J01122-A
• Cotransformed J01122/J01129 into TGI cells in order to determine relative RFP flouresence levels. Our assay includes untransformed TGI cells, J01122 transformed, J01129 transformed, and J01122/J01129 cotransformed cells.
• KAITLIN GOT HER BADGE! AND COMPLETED HER SAFTEY TRAINING!
• Finished ligating and subcloning 994 Library into Gene Hogs and plated.
• Kaitlin started to make pSBdXS construct described in the construction file. Kaitlin will set up ligation and Dan will transform it into TGI cells.

KAD_06/22/06
1. We grew cultures of pSB3C6-J01022, pSB3C6-J01122-A, pSB3C6-J01093, and pSB3C6-J01093 (all in TG1 cells) to saturdation.
2. Put samples in tecan
3. Measured the fluorescence at wavelengths appropriate for RFP:

• pSB3C6-J01122; at saturation: 20, at midlog: 31
• pSB3C6-J01022; at saturation: 2002, at midlog: 267
• pSB3C6-J01093: at saturation: 24, at midlog: 19
• pSB3C6-J01122/pSBIA2-J01129; at saturation: 2484, at midlog: 311

We did this experiment to test our lock3 and key3. In order to do this, we had to compare results with this test of cells with RFP only (pSB3C6-J01122), the lock only (pSB3C6-J01122), both the lock and the key (pSB3C6-J01122/pSBIA2-J01129), and cells containing no RFP gene (pSB3C6-J01093).

The results from these experiments seem too good to be true. The only way this could be wrong is if we in reality cotransformed J01022 (OnRFP) with the key plasmid instead of the lock3 plasmid, J01122. To resolve this, we have also decided to sequence our miniprep of pSB3C6-J01122-A to confirm the sequence of the lock reporter.

A second attempt to make the Lib994 key3 variant library failed to generate colonies. Therefore, we tried to figure out why it failed. We ran an analytical gel of the ligation product for the library. We cut the plasmid with AlwnI because this enzyme would theoretically produce two bands (726 and 1509 bp) from the unligated strand and only one band (2235 bp) from the ligated/circular strand. Unfortunetly, the gel produced unexpected results--multiple bands appeared the gel. Apparently what happened was that when we had previously cut with XbaI, only one site was cut, and the sticky ends (in some cases) ligated back together, and then fragments were cut with the AlwnI, producing an array of fragments varying in length.

We've designed a new strategy to make the 994 library. We will do a PCR product off J01129 and do a suffix insertion into pSB1A2-R0040 as described in the modified construction file.

KAD_06/19/06

• Ran analytical gel on PCR products (A (the 994 key3 library) and B (J01093)) made 06/17/06. Visible bands present for each sample confirmed success of PCR.

EIPCR of 994 key3 library

• Digested and recircularized 994 key3 library created by PCR.
• Transformed 994 key3 library into GeneHogs and TGI with pSB3C6-J01122 by electroboration. Plated products.
• Cotransformed J01122 and J01129 by heat shock. Plated product.