Team:WashU StLouis/Notebook

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Notebook

Reading about labwork is almost as fun as doing labwork

June
1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30
June 1st


No lab work done



No lab work done

June 2nd


  • First day in lab!
  • Made 1L of LB Media
June 3rd


No lab work done

June 4th


No lab work done

June 5th


No lab work done

June 6th


  • Performed gDNA Extraction on E. Coli MG1655 and Synechocystis 6803
June 7th


  • Designed PCR primers for pgk, pck, fldA, petF, ATP Synthase 1, and ATP Synthase 2
June 8th


No lab work done

June 9th


  • Primers Arrived
  • Ran PCR on ATP Synthase 2 , pgk, fldA, petF, and pdCas9 plasmid
  • Ran all PCR products on gel and recovered ATP Synthase 2, fldA, and petF
  • Ran gradient PCR on pdCas9 plasmid
June 10th


No lab work done

June 11th


No lab work done

June 12th


  • Made and ran gel for pdCas9
  • Ran PCR on ATP Synthase 1, pgk, and pck
  • Performed gel extraction on yesterday’s ATP Synthase 2
  • Ran pck, pgk, and yesterday’s gradient pdCas9 PCR products on gel and recovered all genes
  • Performed gel extraction on pdCas9, fldA, petF
  • Ran PCR on psc101 and psc102 plasmid
  • Ran psc101 and psc102 PCR products on gel - were recovered
  • Ran gradient PCR on ATP Synthase 1
  • Made agar plates (forgot the antibiotic)
  • Ran golden gate one pot assembly for fldA petF genes and the pdCas9 plasmid
June 13th


  • Ran ATP Synthase 1 PCR products on gel - no bands seen
  • Gel Extraction for pgk, pck, psc101, and psc102 plasmid
  • Ran PCR on ATP Synthase 1 (3rd times the charm)
  • Ran PCR products on gel - WE SAW BANDS! ATP SYNTHASE 1 RECOVERED
  • Reran PCR on pgk, pck, psc101, and psc102 plasmid because we did not get high enough concentration during gel Extraction
  • Made new agar plates (with the chloramphenicol)
  • Transformed the petF, fldA plasmids into competent cells
  • Plated colonies on agar plates and set in incubator
June 14th


  • Checked for colonies (both genes were successful)
  • Picked colonies and incubated overnight
  • Ran yesterday’s PCR products on gel - bands were light but all were recovered
  • Gel Extraction for pgk, pck, psc101, and psc102 plasmid
  • Reran PCR on pgk, pck, psc101, and psc102 plasmid because we did not get high enough concentration during gel extraction
  • Ran PCR products on gel - all were recovered
  • Gel Extraction for pgk, pck, psc101, and psc102 plasmid
June 15th


  • Miniprepped plasmids for fldA, petF
  • Ran sequence PCR on both plasmids
  • Ran PCR product on gel to confirm
  • Clean and Concentrated PCR product
  • Reran PCR on pgk, pck, psc101, and psc102 plasmid because we did not get high enough concentration during gel extraction (for the fourth time)
June 16th


  • Prepared DNA for sequencing
  • Delivered DNA to WashU Med School
  • Added DPnI to psc101 and psc102 gel extract products, incubated for an hour, did Clean and Concentrate, measured conc
  • Ran Golden Gate assembly on pck, pgk, and ATP Synthase
June 17th


  • Electroporation with golden gate products
  • Redid golden gate for pck, pgk, ATP synthase (did not run properly first time)
June 18th


  • Electroporation with better golden gate products
  • Picked colonies for worse golden gate for pck, pgk, ATP synthase
June 19th


  • Miniprep worse ATP, pck, pgk golden gate
  • Seq PCR worse ATP, pck, pgk golden gate
  • Picked colonies for better golden gate for pck, pgk, ATP synthase
June 20th


  • Made frozen stock of better golden gate pck, pgk, ATP synthase
  • Miniprepped better golden gate pck, pgk, ATP synthase (low concentration)
June 21st


  • Ran sequencing PCR for pgk, pck (4 colonies each)
  • Gel Extraction for pgk, pck PCR product (no bands)
June 22nd


  • Ran sequencing PCR for pck and pgk on a gradient
  • Gel Extraction for pck PCR product was successful, not pgk
June 23rd


  • Ran sequencing PCR for pgk on a different gradient
  • Gel Extraction for pgk PCR product was not successful
June 24th


No lab work done

June 25th


  • Picked four new colonies of pgk
  • Started 4 new cultures from frozen stock
June 26th


  • Made frozen stock of the 4 new colonies
  • Miniprepped all 8 pgk cultures and measured concentrations
  • Ran gradient PCR on all 8 pgk plasmids
  • Prepped and sent ATP synthase and pck in for sequencing
June 27th


No lab work done

June 28th


  • Mixed new pgk sequencing primers
June 29th


  • Ran gradient PCR on all 8 pgk plasmids
June 30th


No lab work done

July 1st


No lab work done

July 2nd


No lab work done

July 3rd


4th of July!

July 4th


No lab work done

July 5th


  • Sequencing results for ATP and pck were not good
  • Reran seq PCR on ATP Synthase (bad results)
  • Picked new ATP colonies
July 6th


  • Miniprepped overnight cultures of ATP 5-8 and forgot to make frozen stock
  • Ran PCR on ATP plasmids 5-8
  • PCR for pgk with new primers with gradient
  • Ran pgk PCR product on gels
  • Ran golden gate on pgk PCR gel extract
July 7th


  • Electroporation with golden gate pgk product
July 8th


  • Picked 4 pgk products and inoculated them overnight
July 9th


  • Made frozen stock of new 4 pgk cultures
  • Miniprepped cultures
  • Seq PCR on new cultures
  • All 4 cultures showed correct bands
  • Clean and Concentrate pgk PCR product
  • Prepped pgk for sequencing
July 10th


  • Delivered pgk for sequencing
July 11th


  • New ATP synthase sequencing primers and pck regular primers came in
  • Seq PCR of ATP synthase plasmids with new sequencing primers. No bands
  • Picked 4 new ATP synthase colonies (again)
  • Inoculated MG1655 from frozens stock to make more genomic DNA
July 12th


  • Extracted genomic DNA from MG1655
  • Miniprepped new ATP cultures
  • Ran sequencing PCR on new ATP and got no bands at all. Will use newly transformed cells tomorrow
  • Ran PCR with pck primers, got light bands
  • Reran PCR with pck products
July 13th


  • Ran pck on gel
  • Gel extracted pck and combined gel extracts to improve concentration
  • Golden gate ligation on pck
  • Ran ATP sequencing PCR again with different backbone primers
July 14th


  • Ran gel from yesterday’s ATP PCR. No bands
  • Ran PCR for pfo
  • Incubated pck and BioBrick cells
July 15th


  • Miniprepped pck and biobrock genes (HSP)
July 16th


No lab work done

July 17th


  • Ran ATP seq PCR gradient again. No bands. Done with ATP
  • BioBrick digestion, ligation, transformation with HSP and GFP
  • Ran PCR seq for pck and gel extract
July 18th


  • Delivered pck sequence to med school
  • Ligated backbone for BioBrick
  • Extracted more genomic DNA from MG1655
  • Gradient PCR on pfo
  • Received 𝛥aceE cells from Yale
  • Rehydrated and incubated 𝛥aceE cells
July 19th


  • Ran PCR and gel extract for fldA backbone and petF backbone
  • DPn1 the two backbones
  • golden gate ligation on pfo gene and two backbones
  • Streaked colony from 𝛥aceE plate onto LB/kan plate
July 20th


  • Making media for competent cell protocol
  • Made new LB with MgSO4 for chemical transformation
July 21st


  • Digested BioBrick miniprep to check length
July 22nd


  • Chemically competent cell protocol with 𝛥aceE
  • Miniprepped pfo-petF and fldA-petF
  • Seq PCR miniprepped pfo-petF and fldA=petF
  • Remade LB with MgSO4 with correct concentration
July 23rd


No lab work done

July 24th


  • Restarted comp cell protocol (3rd times the charm)
  • Clean and Concentrate junction 2 for pfo-fldA
  • Conducted HSP test in LB
July 25th


  • Redid HSP test in minimal media M9
  • Recultured BioBrick and DH10B control
July 26th


  • Reset up BioBrick experiment but grew diluted cells with LB instead of M9, so we have to do it again
July 27th


  • Redid HSP test in minimal media M9
July 28th


No lab work done

July 29th


No lab work done

July 30th


No lab work done

July 31st


  • Chemical transformation of pfo-petF, pfo-fldA, petF, fldA into knockout strain, plated 80 and 240 ul, also control
  • Part 1 of interlab study (LUDOX)
  • Transforming 5 interlab plasmids
August 1st


  • Picked and incubated electron donor colonies plated yesterday
  • Picked and incubated interlab colonies plated yesterday (2 per construct)
August 2nd


  • Made frozen stock of incubated colonies and interlab parts
  • Redid BioBrick experiment at 37C

August 3rd


No lab work done

August 4th


No lab work done

August 5th


No lab work done

August 6th


  • Started induction but had to stop bc no frozen stock of fldA-pfo or petF-fldA in DH10B
  • Transformed fldA-pfo and petF-pfo into DH10B
August 7th


  • Made frozen stock of 𝛥aceE
  • Made 60% ethanol
  • Incubated 8 constructs for biotin assay w/ methanol extraction (4 constructs and blank in DH10B and 𝛥aceE)
August 8th


  • Methanol extraction assay (grew cells and froze pellets)
August 9th


  • Assayed petF-aceE cells. Absorbance was too low to be meaningful
August 10th


  • Ran preliminary sonication test
  • Incubated large volume of DH10B
August 11th


  • Ran large sonication test on DH10B to decide protocol
August 12th


  • Incubated large volume of DH10B
August 13th


  • More sonication tests
August 14th


  • Made ATP standards from assay instructions
August 15th


  • Made more LB
August 16th


  • Inoculated pgk, pck, DH10B for assay tomorrow
August 17th


  • ATP luminescence assay
  • Inoculated 4 constructs and blank in 𝛥aceE cells for tomorrow
August 18th


  • Nothing grew, couldn’t do induction
August 19th


  • Inoculated DH10B constructs and control from frozen stock
August 20th


  • DH10B electron donor induction, froze pellets, measured OD600
  • Re-transformed constructs into 𝛥aceE cells
August 21st


  • Sonicated and assayed pellets for biotin (1 minute, 40 amp, 1mL PBS)
  • Reinoculated 𝛥aceE constructs in all antibiotic combinations to determine problem, in LB and plates
August 22nd


  • Visited Monsanto
August 23rd


  • Measured absorbance data from biotin in DH10B cells
  • Did ATP Assay and got luminescence data
  • Incubated all four 𝛥aceE electron donor constructs from frozen stock plates and original plates and incubated 𝛥aceE and DH10B
August 24th


  • Nothing grew from yesterday's inoculations
  • Interlab study
August 25th


  • Remade competent cells
  • Finished interlab study (FITC) and sent to iGEM HQ
August 26th


No lab work done

August 27th


No lab work done

August 28th


No lab work done

August 29th


No lab work done

August 30th


No lab work done

August 31st


No lab work done

September 1st


No lab work done

September 2nd


  • Picked colonies from 4 new 𝛥aceE plates and incubated
September 3rd


  • 𝛥aceE cell induction
September 4th


No lab work done

September 5th


No lab work done

September 6th


No lab work done

September 7th


No lab work done

September 8th


No lab work done

September 9th


No lab work done

September 10th


No lab work done

September 11th


No lab work done

September 12th


No lab work done

September 13th


No lab work done

September 14th


No lab work done

September 15th


  • Incubated pck, pgk, DH10B from frozen stock
September 16th


  • Ran ATP assay
  • BioBrick digestion and ligation
  • Sonicated 𝛥aceE pellets
  • Ran 𝛥aceE cell absorbance scan
  • Ran biotin assay on 𝛥aceE cells
September 17th


No lab work done

September 18th


No lab work done

September 19th


No lab work done

September 20th


No lab work done

September 21st


No lab work done

September 22nd


No lab work done

September 23rd


No lab work done

September 24th


No lab work done

September 25th


  • Transformed ligated petF and pck BioBrick plasmids into DH10B (wrong antibiotic)
September 26th


No lab work done

September 27th


  • Transformed ligated petF and pck BioBrick plasmids into DH10B (kan = right)
  • Wrapped pck and pgk plasmids to be sent to Cardiff University iGEM
September 28th


  • Small colonies grew on all plates
  • Picked colonies and inoculated
  • Sent plasmids to Cardiff University iGEM
September 29th


  • Miniprep on pck and petF BioBrick
  • Reusupended TetR-pTet gBlocks
September 30th


  • Digestion/Ligation/Transformation of pSB1C3 and tetr-ptet BioBrick
October 1st


  • TetR-pTet colonies grew, picked colonies
October 2nd


  • Miniprepped TetR-pTet BioBrick
October 3rd


  • digested BioBricks to check bands sizes
October 4th


No lab work done

October 5th


No lab work done

October 6th


No lab work done

October 7th


No lab work done

October 8th


No lab work done

October 9th


No lab work done

October 10th


No lab work done

October 11th


  • Ran PCR on BioBrick plasmids
October 12th


  • Ran PCR products on gel
  • Only petF showed up
October 13th


  • digestion of pck, TetR-pTet, and pSB1C3 with EcoRI
October 14th


  • Digestion of pck, TetR-pTet, and pSB1C3 with PstI
  • Ligated pck and TetR-pTet to pSB1C3
  • Transformed into DH10B
October 15th


  • Only TetR-pTet grew
  • Ran colony PCR with 8 pck colonies and 8 TetR-pTet colonies
October 16th


  • Ran colony PCR products on a gel but got no bands
  • Miniprepped pTet1-8
  • Ran PCR on pTet 1-8 plasmids to check if insert was present
  • Ran PCR product on gel, got the band!
  • Prepped petF, pck, and TetR-pTet to be sent to iGEM HQ
October 17th


  • Last day in lab!
  • Sent parts to iGEM!
October 18th


WIKIFREEZE

October 19th


Done in lab

October 20th


Prepping for Jamboree

October 21st


Prepping for Jamboree

October 22nd


Prepping for Jamboree

October 23rd


Prepping for Jamboree

October 24th


Prepping for Jamboree

October 25th


Prepping for Jamboree

October 26th


Fly to Boston for Jamboree

October 27th


GIANT JAMBOREE

October 28th


GIANT JAMBOREE

October 29th


PRESENTATION

October 30th


Fly back to St. Louis

October 31st