Berk2006-LocksAndKeysTeam

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

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.