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From 2012.igem.org

Contents 1 6/6/12 2 6/7/12 3 6/8/12 4 6/9/12 5 6/11/12-6/16/12 6 6/18/12 7 6/19/12 8 6/20/12 9 6/21/12 10 6/22/12 11 6/25/12 12 6/26/12 13 6/27/12 14 6/28/12 15 6/29/12 16 7/2/12 17 7/3/12 18 7/5/12 19 7/6/12 20 7/9/12 21 7/10/12 22 7/11/12 23 7/12/12 24 7/13/12 25 7/14/12 26 7/15/12 27 7/16/12 28 7/17/12

6/6/12

• Set up equipment
• Autoclaved 2x 1L Sterile ddH₂O
• Autoclaved 500 mL LB Broth for scale up of luxS (Plate 3 Well 2H, pSB1A2), plsr (Plate 2 Well 14H in pSB1A2 resistance to Amp)
• Added iGEM logo to wiki

6/7/12

• Resuspended available biobricks luxS & plsr (resuspended in 10uL ddH₂O)
• Resuspended DNA transformed into DH5α (40uL DH5α+2uL DNA) & plated on LB plates w/ 15 uL of 100 mg/mL Amp spread on surface
• Requested lsrR & lsrK from iGEM HQ

6/8/12

• iGEM Wiki now split into two separate Notebooks
• Plenty of colonies, too many to count(TMTC) grew from plsr & luxS transformation from 6/7/12
  • Selected one colony each
  • Grew in 10 mL LB+Amp (100 ug/mL)
• Contacted researchers for V. harveyi bioassay & lysostaphin, V. harveyi must be purchased through ATCC ($300), lysostaphin obtained thru MTA.

6/9/12

• ~2mL of LB evaporated during incubation
• Cells spun down @ 5000 rpm for 10 min
• Miniprepped 4x Lysis into 1 column
• [plsr]=112 ng/uL
• [luxS]=114 ng/uL

6/11/12-6/16/12

• Dry lab work

6/18/12

• Obtained & resuspended primers for eGFP & plsr

6/19/12

• performed PCR w/ NEB standard taq kit w/ primers for eGFP & plsr
• Tested 2 annealing temperatures, 55C and 50C
• Ran gel w/ PCR product (2% agarose), saw clear eGFP bands, but possible .1kb obscured by loading dye.

6/20/12

• Performed qiagen PCR purification & nanodropped (it like it's hot)

6/21/12

• Ran gel of yesterday's PCR purification

• • EtBr Gel 2% Agarose
    • Lane 1: NEB 2-log ladder (1 ug loaded)
    • Lane 2: PCR of eGFP using plsr_XhoI Fwd & eGFP_EcoRI Rev primers w/ 55C annealing temp.
    • Lane 3: PCR of plsr using plsr_BglII Fwd & plsr_XhoI Rev primers w/ 55C annealing temp.
    • Lane 4: PCR of eGFP using plsr_XhoI Fwd & eGFP_EcoRI Rev primers w/ 50C annealing temp.
    • Lane 5: PCR of plsr using plsr_BglII Fwd & plsr_XhoI Rev primers w/ 50C annealing temp.
    • Lane6/7: Null
    • Lane 8: 1kb ladder left at rt o/n
• Obtained & transformed pET-26b

6/22/12

• Miniprepped pet26b and digested with BglII and EcorR

6/25/12

• Ran gel of eGFP/plsr ligation
• Ordered restriction enzymes from the Cell Center

6/26/12

• Attempted Gel Purification of digest products from 2/25/12
  • Yields for Gel purification were too low to be useful (>1 ng/uL)
• Digested purified PCR rxns (annealing temperature=55C)

6/27/12

Ran gel of digested PCR rxns (55C), found no evidence of successful digestion.

6/28/12

Purified digest and attempted

6/29/12

• Performed digest of pET-26b, eGFP, and plsr
• Spread Biobrick shipment on Amp plates (lsrR, lsrK)

7/2/12

• Selected colonies from lsrK and lsrR streaked plates
  • Grew in 10mL TB Broth

7/3/12

• Ran gel of ligated product, no size shift, unlikely that ligation was successful.
• Ligation rxn transformed to make sure

7/5/12

• Ligation transformation of pET-26-plsr-GFP failed, no colonies present
• Talked to Dan:
  • When using primers to introduce restriction sites into

7/6/12

• Redesigned primers for plsr & egfp
• Set up TC Incubator & hood
• Contacted people in the FDA thru personal contacts for human practices

7/9/12

• Made Inactivating Solution for restriction digest
  • 50mM EDTA (pH=8.0)
  • 50% Glycerol
• Planned test restriction digest pET-26b troubleshooting tomorrow
• Trained new users for Tissue Culture

7/10/12

• Troubleshooting of pET-26 digestion
  • Digest of pET-26b w/ BglII and XhoI
  • Time course:
  • 10 min
  • 30 min
  • 3 hr
  • 6 hr
  • o/n
  • Inactivate w/ Inactivating Solution (10uL/50uL of restriction reaction)
• Nikita, Ashwin, Avin all have acceptable sterile practice
• Resuspended IDT luxS synthesized DNA
• Transformed synthesized luxS into DH5a

7/11/12

• Ran 2% gel of digest, did not see signs of any restriction
• Re-planned pET-26b digest troubleshooting
• Selected luxS colonies

7/12/12

• Thawed out 293T-CMV-Gaussia cells from Sarkar LN2
• Miniprepped luxS colony, digested w/ BamHI-HF & EcoRI-HF
• Performed gel extraction of luxS fragment

7/13/12

• Passaged 293T-CMV-Gaussia cells 1:10 & 1:5 for weekend
• Ligation of luxS w/ pET-26b
• Ligation rxn transformed into DH5a

7/14/12

• Ligation showed successful transformants

7/15/12

• Colonies selected by Mike, grown in 10mL LB

7/16/12

• SAAST Presentation
• Miniprep of 10mL pET-26b-luxS cultures
• Ran gel of minipreps from 3 pET-26b-luxS colonies to confirm successful ligation
• pET-26b-luxS #1/#2 are slightly larger, indicating successful ligation, not as sure for pET-26b-luxS #3
• Last lane is unligated pET-26b vector

7/17/12

• Ran gel to determine if insertion of pET-26b-luxS was correctly ligated
  • Gel showed there was a possible size shift corresponding to insertion in all three selected colonies of pET-26b-luxS colonies

• • From left to right (optimum temperatures bolded): Ladder, plsr @45C,52C,58C,65C, egfp @52C,58C,65C
• Performed 100uL PCR of plsr @65 & 100uL PCR of egfp @58 for further ligation experiments.