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
