Berk2006-LocksAndKeysTeam

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.