Team:Brown-Stanford/Lab/Notebook/Week10

August 15, 2011

PowerCell

• Ligation of bricks:Ana, CscB
• Transformed
• Digest remaining Ana and CscB (from gel extracted stuff)
• GIGANTIC PCR:
• Ones that have worked in the past: pSB1C3 (8/10 Phu 40-50C), Ana (8/12 Phu 47C), CscB (8/12- CN primers, 47C Phu), Nos (see 8/8 Taq 45-55C), GFP (see 8/8 Taq 45-55C)
• We will do: pSB1C3, Ana, CscB, GFP (45-55C, Phu); 4 temps, 60ul each
• Ran GIGANTIC PCR products on gel; each template gets its own small gel (30ul product each lane, so eight lanes total);
• CscB starts with 1kb ladder, 55-45;
• pSB1C3 starts blank, 1kb ladder, 45-55
• Ana starts 100bp ladder, 45-55
• GFP starts 100bp, 45-55
• Got sequence of Gibson miniprep back - it’s contamination, not our sequence.
• transformation:
• transformation of 25 into 623 was successful!!!!!
• picked 623+25 into liquid chlor+kan liquid LB
• started large 623 culture in lb + chlor for making competent
• made neo50, neo75, neo100 plates for selection testing
• anabaena will be plated on nitrocellulose and straight on the bg11.
• 623+25, 443 to liquid LB culture in preparation for transformation tomorrow.

REGOBricks

• Gel extraction of construct from successful and first PCR
  • Extremely low yield at 1.7 ng/μL. Will try increasing washes and time spent using the gel extraction protocol.
  • Second PCR, this time taking 4 lanes instead of the single lane we took in the first. Changes to protocol: instead of running the first QG suspension through the filter once, it was run 3 times through; Elutions with EB to bring the DNA back out to solution were performed slowly and repeatedly, as the buffer was allowed to settle for 5 minutes and then pushed through three times.
  • Yields in two vials (needed to split the protocol up since the amount of gel harvested was greater than a single filter would allow) were 3.9 and 4.3 ng/μL, representing a small increase in yield.

• Plasmid Prep of pUB110: using modified qiagen protocol w/ lysozyme www.qiagen.com/literature/render.aspx?id=467
  • Protocol calls for prep at OD 0.8-1.2, but this culture of B. subtilis was grown over the weekend, OD ~ 1.5
  • Growth > 16 hrs - bad, because cells begin to lyse.

• Anyways, will attempt plasmid prep
• Results of Plasmid Prep: Two tubes of highly concentrated DNA, stored in biocementation gel running things box

• Tube 1: 360 ng/ul pUB110
• Tube 2: 330 ng/ul pUB110

• Investigation in making various media needed for protoplast transformation for tomorrow:

• Reagents:

• 1x Spizizen w/ 18% glycerol (have)- CAN AUTOCLAVE
• 18 ml glycerol
• 2.31 g Spizizen’s minimal salts
• Dissolve in distilled water, final volume 100 ml
• Autoclave

• 2x SMM (do not have)- FILTER STERILIZE
• For 1000 ml:
• Sucrose 342.3 g
• Maleic Acid 4.64 g
• MgCl2 8.13 g
• pH to 6.5

• 4x PAB (have, dark-caramel colored, remake) - FILTER STERILIZE- MADE
• For 1 L- for 4x add only 250 mL H20.
• 10 g Peptone
• 1.5 g YE
• 1.5 g Beef Extract
• 3.5 g NaCl
• 1.0 g Glucose
• 3.68 g KH2PO4 (dibasic)
• 1.32 g K2HPO4 (monobasic)

• SMMP (do not have) - FILTER STERILIZE - WILL MAKE TOMORROW MORNING (WMTM)
• Aseptically add 4x PAB to 2x SMM.
• Filter Sterilize in 110 mL aliquot.

• SMMP + BSA (have, dark-caramel colored, remake) - FILTER STERILIZE (WMTM)
• Place 110 ml SMMP in 600 ml beaker.
• Add 1 g BSA to surface. Do not stir or swirl. Let it slowly dissolve. Come back after an hour, gently swirl, filter sterilize.

• 40% PEG (have)

• vy-R5 Regeneration media (have, dark-caramel colored, remake)- MADE
• 41.2 g Sucrose
• 4.0 g MgCl2
• 4.0 g Glucose
• 0.1 g K2SO4
• 0.08 g Casamino acids
• 2.6 g MOPS
• 2.4 g Proline
• 4.0 g YE
• Add 80 ml water, filter sterilize.

• 8.0 g Difco agar = 300 ml

• Add 380 ml water, filter sterilize. When cooled, add following sterile ingredients (* = located in stock concentrations in 378 back room)

• 1.0 ml of 2% KH2PO4
• 2.2 ml of 40% CaCl2 - 2H2O
• 1.32 ml 5 M NaOH

August 16, 2011

PowerCell

• ligation and transformation of Ana and CscB = failed, again. sadface.
• second transformation with Ana and CscB ligation product
• another 12μl of each
• ran with +control (GFP from registry)
• used 1μl (conc ~1200 ng/ml!)
• just realized it’s hella early in the day to transform, but stuck them in the incubator early anyway because there’s really no threat of satellite colonies judging from our success rate...
• Transformation of BBa_J45200 (banananananas) and BBa_K325909 (LuxBrick, pBAD control only requires induction by arabinose)
• Did GIGANTIC GEL EXTRACTION MEGA PCR. Was successful; we have UNCOMFORTABLY LARGE amounts of linear biobricks in the 4C
• Digestions: EP digested Ana (33n/u), CscB (20n/u), and pSB1C3 (25n/u)
• 25ul rxn: with 0.5ul of each RE and 22.25ul of DNA
• 1hr incubation at 37C, heatkill enzyme at 80C for 20min
• Ligations (on just Ana and CscB):
• tried 1:1, 3:1, 7:1 insert/vector ratios in parallel, 10ul rxn, 0.5ul of T4 ligase
• used 50ng of vector, added various amounts of digested Ana and CscB as necessary
• had to make the 7:1 CscB rxn at 18ul due to concentrations
• let reaction sit at room temp for 30min before transforming 3ul into 100ul of TOP10
• plated 400ul of cells
• put remaining 7ul into 16C for overnight, will transform again tomorrow morning
• transformation:
• lei and jovian are in charge of making 623 competent for jesse’s future use
• transform anabaena with 443, 623+25. This should goddamn work.
• there was some black stuff in the e. coli pellet...contamination?
• same-plate control of anabaena serial dilution
• check on neo gradient experiment
• started pRL25 for plasmid prepping (several tubes!)

REGOBricks

Transformation of S. pasteurii, a repeat experiment of our first failed transformation since we were unsure of the quality of our Kan-recovery plates.

August 17, 2011

PowerCell

• Got one transformant on cscB from two days ago! Small number of transformants on Luxbrick
• liquid cultured these in preparation for miniprep
• Transformed 3ul of the remaining Ana and CscB ligation reactions (were in 16C overnight, then stored at -20C until late afternoon)
• 30min on ice, 45sec at 42C, 1hr recovery in rotisserie; plated on LB chlor
• retransform positive controls (banana), on AMP plates this time. Did serial dilutions to get an idea of how competent our cells are--1x, 10^-1x, 10^-2x.
• Do verification PCR of ligation
• VF2, VR
• confusing - bands appear to be in the right places, but it’s as if everything got mixed - Ana has both the bands you’d expect to see for Ana and for cscb. Cscb same story.
• Cut out bands - could send in for sequencing? Is concentration high enough?
• Liquid prep
• plasmid prep prl25, prl25 + 623
• transformation
• transfer conjugation filters to neomycin plates--one on neo50, one on neo100.
• created control plates with five orders of dilution on BG11 without antibiotics

REGOBricks

• PCR of puB11 with biobrick prefix and suffix w/ gradient 50-50C
• used 1.2 uL DMSO as was found most effective in previus PCR trials
• Made cryo-stocks of competent cells for protoplast transformation

August 18, 2011

PowerCell

• Sent small and large bands from both Anabaena and cscB (of verification PCR imaged on 8/17) to elim for sequencing; sequences arrived 8/19
• miniprep of CscB and Lux cultures, also innoculated 10mL cultures
• EP digest of banana smell positive control plasmid--yielded two extremely large bands--several kb larger than expected.

August 19, 2011

PowerCell

• Cryostocks of lux and potential CscB biobricks
• but still no glowing e coli :(
• Elim sequences returned, of the gel extracted bands from verification PCR of possible Ana and cscB ligations (done on 8/17, sent off 8/18). Sequences do not align with any of our expected parts;
• this tells us that the VF2 and VR primers did not amplify our desired insert when we PCRed, the correct insert is not ligated into pSB1C3 :(
• Quick and dirty from scratch PCR.
• re-do PCR of Ana, cscb, backbone
• imaged: Ana, backbone turned out clean, cscB showed three bands of amplification?
• Did an X S digest on pos control plasmid; against uncut plasmid, showed two smaller bands

FRETSensor

• SDS gel, 15uL of each sample
  • 1 - Doc-CFP
  • 2 - Doc-YFP
  • 3 - Coh-Coh
  • 4 - YFP-Doc

August 20, 2011

PowerCell

• Continue the from-scratch/TOPO workflow:
• PCR cleanup (no gel extraction)
• the qiagen PCR cleanup kit destroys yield; Nanodrop readings: Ana 7.8, CscB 11, pSB1C3 8.0
• digestion with EP again
• ~30ul rxn, buffer 3, 2hr incubation at 37, 20min at 80 to heat inactivate
• ligation with backbone
• transformation