Team:Sao Carlos-Brazil/Notebook

Project Notebook

JANUARY

Week 0

  • Proof of Concept Experiment: Irradiation of desiccated yeasts with Exophiala sp. Melanin with a radiometer

JULY

Week 1 (7.11 – 7.17)

  • BioBricks resuspension (BBa_K1499201 and BBa_K081005)
  • Competent E. coli TOP 10 making
  • Competent E. coli TOP 10 test
  • Transformation of previous BioBricks into E. coli TOP 10 chemically competent cells
  • Technical visit to our sponsor Fermentec in Piracicaba
  • Collection of S. cerevisiae strain PE-2 provided by Fermentec

Week 2 (7.18 – 7.25)

  • Second transformation of BBa_K081005
  • Glycerinate of E. coli TOP 10 transformed with BBa_K1499201
  • E. coli TOP 10 transformed with BBa_K1499201 miniprep
  • Attendance at RenovAÇÃO, the annual gathering of Fermentec’s client sugar-alcohol Power Plants at Ribeirão Preto
  • Presentation of our project to Fermentec’s clients and guests at RenovAÇÃO

Week 3 (7.26 – 7.31)

  • Glycerinate of E. coli TOP 10 transformed with BBa_K081005
  • E. coli TOP 10 transformed with BBa_K081005 miniprep

AUGUST

Week 4 (8.01 – 8.07)

  • Several attempts at digesting both BioBricks with restriction enzymes
  • Digestion of both BioBricks with restriction enzymes
  • Attempts at ligating both BioBricks

Week 5 (8.08 – 8.14)

  • Attempts at transforming the ligated BioBricks
  • Resuspension of fragments provided by Twist Bioscience and adapters removal via PCR
  • Attempts at ligating both BioBricks
  • Resuspension and amplification of URA gene deletion cassette gBlock provided by IDT

Week 6 (8.15 – 8.21)

  • Ligation of both BioBricks
  • Attempt at joining two gBlocks with Gibson Assembly
  • Preparation of yeast transformation solutions and reagents

Week 7 (8.22 – 8.28)

  • Successful transformation of ligated BioBricks into E. coli TOP 10 chemically competent cells
  • Collection of S. cerevisiae haploid strains S288C and FT2751L
  • Amplification of URA gene deletion cassette via PCR
  • Transformation attempt of S. cerevisiae strains PE-2, S288C and FT2751L with URA gene deletion cassette

Week 8 (8.29 – 8.31)

  • E. coli TOP 10 transformed with ligated BioBricks miniprep
  • Glycerinate of E. coli TOP 10 transformed with ligated BioBricks
  • Transformation of ligated BioBricks into E. coli BL21 chemically competent cells
  • Glycerinate of E. coli BL21 transformed with ligated BioBricks
  • New transformation attempt of S. cerevisiae strains PE-2, S288C and FT2751L with URA gene deletion cassette

SEPTEMBER

Week 9 (9.1 – 9.7)

  • Measurement of S. cerevisiae transformation attempt with URA- medium
  • Expression test of ligated BioBricks in E. coli BL21
  • SDS-PAGE with expression test samples
  • Collection of 5-FOA at UNESP Araraquara
  • Transformation of BBa_K081005 into E. coli BL21 chemically competent cells

Week 10 (9.8 – 9.14)

  • UVC Irradiation test of E. coli BL21 with ligated BioBricks in a radiometer
  • SDS-PAGE with expression test samples adjusted for concentration
  • CFU counting of irradiation test Petri dishes
  • Attempts at amplifying Gibson joined fragments via PCR
  • Amplification of URA gene deletion cassette via PCR
  • Transformation attempt of S. cerevisiae strains PE-2 and S288C with URA gene deletion cassette with 5-FOA auxotrophic marker
  • Attempt at performing fusion PCR for two gBlocks

Week 11 (9.15 – 9.21)

  • Measurement of S. cerevisiae transformation attempt with URA- medium
  • New attempt at performing fusion PCR for two gBlocks
  • Glycerinate of E. coli BL21 transformed with BBa_K081005
  • Amplification of URA gene deletion cassette via PCR and concentration in SpeedVac
  • New attempt at joining two gBlocks with Gibson Assembly
  • Another attempt at performing fusion PCR for two gBlocks

Week 12 (9.22 – 9.28)

  • New amplification of URA gene deletion cassette via PCR and concentration in SpeedVac
  • Transformation attempt of S. cerevisiae strains PE-2 and S288C with URA gene deletion cassette with 5-FOA auxotrophic marker using a different protocol
  • Attempts at joining several gBlocks separately using fusion PCR
  • Preparation of non-modified S. cerevisiae strains PE-2, S288C and FT2751L for our stratospheric balloon flight
  • High altitude balloon flight carrying our samples
  • Resuspension and cultivation of post-flight samples in solid YM medium

Week 13 (9.29 – 9.30)

  • CFU counting of post-flight samples

OCTOBER

Week 14 (10.1 – 10.7)

  • Two Transformation attempts of S. cerevisiae strain S288C with URA gene deletion cassette with 5-FOA auxotrophic marker
  • Amplification PCR of two different gBlocks
  • Measurement of transformation attempts

Week 15 (10.8 – 10.14)

  • PCR of a gBLock using a tailed forward primer
  • Irradiation of BL21 E. coli with a crosslinker
  • Fusion PCR of two Twist fragments to obtain the complete CDS of S. marcescens nucA nuclease
  • Irradiation of BL21 E. coli with a radiometer
  • Transformation of S. cerevisiae strain S288C with URA gene deletion cassette with 5-FOA auxotrophic marker using an electroporation protocol
  • Amplification of fusion PCR product (nucA)
  • Taq incubation treatment to add hangAs to the fragment
  • Ligation attempt of nucA product in pGEm T Easy Vector
  • Digestion of nucA product with BamHI and XhoI
  • Ligation attempt of nucA product in pET-28a Vector
  • Transformation attempt of S. cerevisiae strain S288C with URA gene deletion cassette with 5-FOA auxotrophic marker
  • Streak plates of possible positive yeasts colonies in 5-FOA selective medium

Week 16 (10.15 – 10.21)

  • Confirmation PCR of URA gene deletion
  • Nested fusion PCR of two gBlocks
  • UVC Irradiation in laminar flow hood of S288C with melanin
  • CFU counting of S288C of irradiation test Petri dishes
  • UVC Irradiation test of E. coli BL21 and E. coli TOP10 with ligated BioBricks in a laminar flow hood
  • CFU counting of BL21 and TOP10 of irradiation test Petri dishes
  • Confirmation PCR amplicons digestion with NcoI