Team:Virginia/Notebook

Safety →
Lab work:
  • Shelf organization: Acids, Proteins, Buffers, Gels, Agarose, Salts, Carbs
  • Competent Cell Formation (K12)
 Agenda:
  • PrG Determination (1-4)
  • Orthogonality Research
  • Competent Cell Formation Cont. (K12)
  • Growing K12 cells and making buffer (sterilized transformation buffer)
    • Plans to grow another batch with XL1 Blue tomorrow
Modeling:
  • Research docking software and test
Lab work:
  • Competent cells: Beginning competent cell preparation for E. coli XL1-Blue and K-12
 Lab work:
  • Continued preparing competent cells
Protecting group:
  • Phenyl acetyl protecting group, cleaved by penicillin g acylase
  • Truncated from 5 PGs to 3
  • 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
Funding
  • Anders finished half application for grant
 Lab work:
  • Continuing competent cell preparation
 Lab work:
  • 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 grown in overnight liquid culture at 37°C shaking
 Lab work:
  • JW cells frozen in glycerol stock at -80°C (5 tubes)
 Lab work:
  • Prepared growth media for pr-leu uptake test
 Lab work:
  • Began pr-leu uptake test
  • Test for lawn growth or individual colonies on a spread plate (for CRISPR)
 Lab work:
  • Took OD measurements for uptake test
  • CRISPR plate - lawn produced
 Lab work:
  • Pr-leu uptake 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 Lab work:
  • Pr-leu uptake test failed again, cells did not lyse
  • Started a new pr-leu uptake procedure
Wiki team meeting - wiki design Lab work:
  • 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 of transformed bacteria prepared for glycerol storage
 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°C freezer
 Lab work:
  • Second sample taken from enzyme reaction and frozen in -20°C 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 came out as expected - No growth on plates with pr-leu, but growth present on plates supplemented with leucine
  • Enzyme test was inconclusive due to poor sensitivity of the LC/MS - Suspected due to buffer contamination
 Lab work:
  • PCR performed on LeuS gene, product was purified
 Lab work:
  • Confirmatory digest performed on LeuS PCR product
  • XL1-blue cells transformed with LeuS-ampR plasmid - plated and incubated at 10:45pm
 Lab work:
  • Excessive growth on control transformation plate from 7/5 - Ligation likely did not succeed
  • Inoculated precultures of LeuS and terminator transformed cells for miniprep and sequencing
 Lab work:
  • Redoing enzyme test (again) with a new protocol
  • Performed a miniprep to obtain LeuS and terminator plasmids
 Lab work:
  • Sent off minipreped LeuS and terminator plasmid DNA for sequencing
  • Performed confirmatory digests on LeuS and terminator plasmid DNA
 Lab work:
  • Sequencing of LeuS and terminator plasmid failed due to poor quality of DNA samples
  • Performed PCR and PCR purification on E. coli genomic DNA to obtain LeuS gene
 Lab work:
  • Talked with Professor Kozminski about PCR
  • Set up another PCR reaction and PCR purification to obtain LeuS gene
  • Transformed XL1-blue cells with terminator plasmid
 Lab work:
  • No colonies grew on the terminator transformation plate
  • Restarted enzyme efficacy test
  • Transformed XL1-blue cells with RFP, T1, and T2 plasmids
 Lab work:
  • Removed and stored a sample of the enzyme test
  • Observed growth on the RFP, T1, and T2 plates, no growth on control
  • Started precultures with colonies from the terminator 1 and 2 plates
  • Performed PCR on LeuS gene (genomic DNA)
 Lab work:
  • Performed gel electrophoresis on LeuS PCR product, obtained wrong product
  • Extracted DNA from gel
  • Performed minipreps
 Lab work:
  • Confirmatory digest of minipreps from 7/15
  • Performed a ligation reaction for T1 and T2
 Lab work:
  • Transformed XL1-blue cells with T1, T2, and an RFP control
  • Extracted digested products from agarose gel
  • Plated again using transformed cells from 12am at 1:30pm
  • Transformed and plated new cells at 3:30pm
  • Ligation reaction performed for T1 and T2 using DNA extracted from gel
 Lab work:
  • Transformed XL1-blue cells with the 2 ligation products (T1 with PCR, T2 with PCR)
  • Began a preculture of XL1-blue cas9 cells in LB+cam at 5:45pm
 Lab work:
  • Performed a restriction digest
  • Cells transformed and incubated at 37°C at 9:45am
  • T1, T2, RFP, and control removed and plated at 11:15am
  • Started uptake and enzyme activity tests with N-methoxy-leu
 Lab work:
  • Continued performing uptake test for N-methoxy-leu
 Lab work:
  • Continued performing uptake test for N-methoxy-leu - lysed cells and stored cell lysate
  • Performed minipreps and confirmatory digests - No result from digests because no EtBr was in the gel
 Lab work:
  • Redoing digests from 7/21 and running a new gel
  • Received sgRNA from biobasic, prepared and stored the sgRNA at -20°C
  • Digested CRISPR RNA insert and Cas9 vector
 Lab work:
  • Performed digests with BSA1 on the CRISPR vector and insert
  • Performed a ligation reaction between the vector and insert
 Lab work:
  • Realized that the primers used to PCR the LeuS gene were incorrect - Therefore, our T1 and T2 biobricks did not contain the correct DNA sequences
 Lab work:
  • Continued the PrG uptake test - purification of the cell lysate
  • Performed a miniprep for the T1 and T2 terminators (for biobricking)
 Lab work:
  • Transformed and plated KanR gene from kit
  • Streaked pS1M27 cells from gel stab to Tet plate
 Lab work:
  • Re-transformed and plated KanR gene on CAM plates
  • Re-streaked pS1M27 plate
 Lab work:
  • Miniprep performed for pS1M27
  • Re-transformed KanR DNA
  • Performed a PCR reaction to obtain LeuS gene from genomic DNA
 Lab work:
  • Confirmatory digest performed on PCR product (LeuS gene)
  • Restriction digest for biobrick #1
  • Performed ligation reactions between the LeuS gene and the T1 and T2 genes
 Lab work:
  • Performed a ligation reaction between CRISPR Cas9 genes and sgRNA sequence
  • Minipreped ZeoR, KanR, T1, and T2 plasmids
  • Confirmatory digests performed for T1 and T2 plasmids
  • Performed a ligation reaction with T1 and T2 genes, transformed products
 Lab work:
  • PCR performed to confirm CRISPR/Cas9 and sgRNA gene ligation
 Lab work:
  • Digests performed to redo LeuS and terminator ligation
 Lab work:
  • Ligation reaction performed
  • Received IDT DNA: Penicillin G Acylase pieces 1 and 2
  • Sequencing of Cas9 plasmid
  • Digested PCRed LeuS product
  • Transformed cells with T1 LeuS lig and T2 LeuS lig
  • Began preparing competent JW strain E. coli cells
 Lab work:
  • Restriction digest of pac1, pac2, kan, and zeo resistance genes
  • Restriction digest of terminators followed by cipping
  • Terminator digest Ex + Sip gel
  • LeuS PCR purification
 Lab work:
  • Performed transformations using the following genes:
    • Promoter from iGEM distribution kit
    • RFP for cell competency check
    • T1-LeuS ligation reaction products
    • T2-LeuS ligation reaction products
 Lab work:
  • Restriction digests on pac1 and pac2
  • Ligation reaction for pac1-pac2-terminator
 Lab work:
  • Miniprepped 9 terminator-LeuS ligation plasmids
  • Transformed pac-terminator ligation
  • Precultured promoter into pac vector
 Lab work:
  • Minipreped Pro1 and Pro2
 Lab work:
  • Performed PCR on CRISPR plasmid
  • Performed a restriction digest to confirm LeuS-terminator plasmid
  • Performed a PCR purification
 Lab work:
  • Performed restriction digests on terminator 1, terminator 2, LeuS from PCR, Cas9 plasmid, and on sgRNA insert
  • Performed sequencing on CRISPR insert
  • Miniprepped 5 pac-term
  • PCR of LeuS gene
 Lab work:
  • Completed PCR purification
  • Performed ligation on zeo and piece E
  • Performed a transformation of zeo-E-tet
 Lab work:
  • Restriction digest of LeuS
  • Redoing competent JW cells
 Lab work:
  • Took nanodrop concentrations of minipreppred ligation reaction
  • Restriction digests
 Lab work:
  • Made primer solutions
  • SDM of Pst 1
  • Transformation and sequencing
 Lab work:
  • Performed digests of T1
  • Digest PCR LeuS
  • Ligated T1 and LeuS
  • Transformed cells
 Lab work:
  • Preculture of term-leuS lig
  • 3 colonies of 6:1 ligation and 1 colony of 8:1 ligation
 Lab work:
  • Miniprep of leuS term ligation
 Lab work:
  • Ran a gel
 Lab work:
  • Performed PCR
  • Performed transformation
Lab work:
  • Preculture of Pst sdm
 Lab work:
  • Performed miniprep
  • Ran confirmatory digest
 Lab work:
  • Performed restriction digests
 Lab work:
  • Performed ligations
 Lab work:
  • Preculture for transformations
 Lab work:
  • 3A assembly of zeo-E-Kan backbone
 Lab work:
  • Sequencing
 Lab work:
  • Performed restriction digest
  • Performed ligation
 Lab work:
  • Performed restriction digest
  • Performed ligation
 Lab work:
  • Nanodrop used to determine concentrations
 Lab work:
  • Nanodrop to determine concentrations and sequencing performed
 Lab work:
  • Ran term and enzyme gels
  • Digest of term performed
 Lab work:
  • Miniprep of term and pro
  • Performed digest
 Lab work:
  • Digest of terminator
  • Success! Got term in lane 5
  • Ligation performed
 Lab work:
  • PCR of mutant LeuS
 Lab work:
  • PCR purifed mut LeuS
  • Digest performed
  • Ligations performed
 Lab work:
  • Miniprep x 27
  • Digest MT 7.1 → CIP
  • Digest piece A and controls
  • Ligations of mut term and piece A performed
 Lab work:
  • Miniprep of HA and mut and term
  • Restriction digest of enz-term and promoter
  • Preculture JW cells
 Lab work:
  • Competent cell procedure for JW
  • Confirmation test followed by plating
 Lab work:
  • Restriction digest
 Lab work:
  • JW5807 competent cell protocol
  • Miniprepped promoter-enzyme-terminator plasmid
 Lab work:
  • Confirmatory digest of promoter-enzyme-terminator plasmid miniprepped on 10/9
  • Sequencing of promoter-enzyme-terminator plasmid miniprepped on 10/9
  • Made minimal medium plates with nothing, 3mM leucine, or 3mM CBZ-leu
 Lab work:
  • digest, gel, ligation of parts into CAM backbones
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