Team:Virginia/Notebook

NOTEBOOK

  • Shelf organization: Acids, Proteins, Buffers, Gels, Agarose, Salts, Carbs
  • Competent Cell Formation (K12)
    • See comp. cell prep protocol in VA iGEM 2016 Lab Protocols
 Agenda:
  • PrG Determination (1-4)
  • Orthogonality Research
  • Competent Cell Formation Cont. (K12)
  • Growing K12 cells and make buffer (sterilized transformation buffer)
    • Plans to grow another batch with XL1 Blue tomorrow
Modeling:
  • Research docking software and test
Competent Cells:
  • Making Sterilized Transformation Buffer (500ml)
  • Took out cells at 9:30am
  • Transferred to 20°C at 11:00 PM, pour one of XL1-Blue and K12
 Competent cells:
  • Cultures left 18&degC;
  • 11pm tonight: into LB + back into 18°C
  • Sterilized filtration buffer
Protecting group:
  • 2?:Phenyl acetyl protecting group, cleaved by penicillin g acylase
  • Truncated from 5 PGs to 3
  • 1: Dipeptide (maybe leu-leu), with a D aa on N-terminus
  • A protease w/ cleavage activity acting on D aa is being researched for increased specificity
Modeling:
  • AD4 returned zero errors w/ synthetase and leucine as ligand
  • Still unsuccessful AG4 and AD4 returned
  • Researching alternatives to direct another approach
  • Unpacked Rosetta ligand modeling
  • IGEM dock successful modeling; concerns about synthetase model
CRISPR
  • Found biobrick to insert plasmid
  • An alternative approach to silencing RNAs and transformation
Funding
  • Anders finished half application for grant
Removing competent cells from 18°C
After 17 hrs:
  • XL1 blue at an OD of 0.012
  • K12 at an OD of 0.16
Put XL1 blue back into 18°C incubator, put K12 into 4°C fridge Competent cells:
  • Removed XL1 blue at a OD of 0.13, centrifuged with buffer, added DMSO, froze with liquid nitrogen
 Lab work:
  • Made CAM plates
  • Made SOC media
  • Competency test
Decided on 4 different PrGs:
  • Cbz leu, pro leu, phenyl acetyl leu, N-methoxy leu
Modeling:
  • Corrected enzyme mistake, now using correct configuration of enzyme
 Lab work:
  • Competency test control plated at 4:50pm
 Lab work:
  • No growth on control plates
 Lab work:
  • JW cells subcultured on LB agar plate in 37°C incubator
 Lab work:
  • JW cells grown in overnight liquid culture at 37°C shaking
 Lab work:
  • JW cells frozen in glycerol stock at -80°C (5 tubes)
 Lab work:
  • Preculture of JW in 20 ml LB
  • Made 50 ml of sterile LB and 0.1g of pro-leu
 Lab work:
  • Began pr-leu uptake test
  • Take OD: 0.07 for each preculture, incubate
  • Test for lawn growth or individual colonies ona spread plate (for CRISPR)
 Lab work:
  • Uptake test OD measurements
    • AM measurements: 1.137 for LB, 1.422 for LB + pro-leu
    • 7 pm measurements:1.104 for LB, 1.994 for LB + pro-leu
  • CRISPR plate - lawn produced
 Pr-leu uptake test:
  • Test failed, bacteria were fixed by ethanol overnight and could not be lysed
  • Restarted test, XL1-blue innoculated in LB at 5:20pm
Policies and Practices:
  • Meeting with Rivanna river waste treatment plant
  • Meeting with open bio labs
 Tested spectrophotometer in the PLSB lab - Not consistent with results in Prof. Kozminski's lab Pr-leu uptake test:
  • Failed again, cells did not lyse
  • Started a new pr-leu uptake procedure
Wiki team meeting - wiki design Created standards for LC-MS Lab work:
  • Transformed XL1-blue with Bba-K1218011 CRISPR Cas9 plasmid
  • Transformed XL1-blue with BBa-B0010 forward terminator plasmid
  • Transformed XL1-blue with RFP control plasmid
 Lab work:
  • Growth on CRISPR, terminator, and RFP plates, no growth on controls
  • Cultures prepared for glycerol storage of transformed bacteria
 Lab work:
  • Optical densities taken for second round of pr-leu uptake and enzyme tests
 Lab work:
  • Optical densities taken again for second round of pr-leu uptake and enzyme tests
 Lab work:
  • Sample taken from enzyme reaction and frozen in -20° freezer
 Lab work:
  • Second sample taken from enzyme reaction and frozen in -20° freezer
 Lab work:
  • Redoing growth uptake test with aerated growth conditions
 Lab work:
  • OD measurements taken for growth test
  • Extracted and purified E. coli genomic DNA via a minikit
  • Began PCR on genomic DNA to obtain the leuS gene DNA
 Lab work:
  • Purified the PCR product
  • Ran a gel to confirm product - bands ran together
  • Took OD measurements for growth test
 Lab work:
  • Extractions performed for enzyme test
  • Redo growth uptake test again
Growth Uptake Test Results
SupplementGrowth at 10:05amGrowth at 4:00pmGrowth at 9:08pm
1 NoneNoneNoneNone
1 NoneNoneNoneNone
3 NoneNoneNoneNone
1 LeuNoneYesYes
2 LeuNoneYesYes
3 LeuNoneYesYes
1 Z 3NoneNoneNone
2 Z 3NoneNoneNone
3 Z 3NoneNoneNone
1 Z 8NoneNoneNone
2 Z 8NoneNoneNone
3 Z 8NoneNoneNone
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