Difference between revisions of "Team:Virginia/Notebook"

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