• Prepared growth media

• Interlab Challenge 2017: for more information, please visit our Interlab site.

• E. Coli DH10B cells made chemically competent

• Golden Gate Assembly of BB1_01, BB1_02, BB1_03, BB1_04, BB1_05, BB1_06, BB1_07, BB1_08

• PCR: 1, 2, 3, 4, 6, 15, 18, 20, 23, 24, 25, 26
Each PCR was loaded on a preparative gel and cut out before undergoing PCR-purification.

• Golden Gate Assembly: BB1_09, BB1_10, BB1_11, BB1_12, BB1_13, BB2_01 (unsuccessful), BB2_02, BB2_03, BB2_04, BB2_05 (unsuccessful)
GG-products are then transformed into competent cells. Colonies with specific antibiotic resistances were selected out by using antibiotic-containing media. The next day, positives clones were confirmed via a OneTaq colony PCR and a gel electrophoresis before a Miniprep was made.

• pSIM5 pre-cultures (DH5alpha + BL21(DE3))

• Transformation of HSM174 + pSIM5 (unsuccessful)

• Yeast S288C gDNA extracted

• PCR: 27, 28
Each PCR was loaded on a preparative gel and cut out before undergoing PCR-purification.

• Golden Gate Assembly: BB1_14, BB1_15, BB2_01 (unsuccessful, even with new BBa_23101 promoter), BB2_05 (unsuccessful), BB2_06, BB2_07, BB2_08, BB2_09, BB2_10, BB2_12, BB2_14, BB2_15, BB2_16, CRISPR (unsuccessful)
GG-products are then transformed into competent cells. Colonies with specific antibiotic resistances were selected out by using antibiotic-containing media. The next day, positives clones were confirmed via a OneTaq colony PCR and a gel electrophoresis before a Miniprep was made.

• Sent for Sequencing: BB1_14, BB1_15, BB2_01, BB2_05, BB2_06, BB2_07, BB2_08, BB2_09, BB2_10, BB2_12

• PCR: 9, 10, 16, 29, 30, 31, 32, 33, 34, 35, 36, 41, 42, 43
Each PCR was loaded on a preparative gel and cut out before undergoing PCR-purification.

• Golden Gate Assembly: BB1_16, BB1_17, BB1_18, BB1_20, BB1_21, BB1_22, BB1_23, BB1_24, BB1_25, BB2_17, BB2_23, BB2_24, BB3_01 (unsuccessful), BB3_02, , BB3_04, BB3_05, BB3_06 (doubtful), BB3_08
GG-products are then transformed into competent cells. Colonies with specific antibiotic resistances were selected out by using antibiotic-containing media. The next day, positives clones were confirmed via a OneTaq colony PCR and a gel electrophoresis before a Miniprep was made.

• Yeast S266C made electro-competent

• Sent for Sequencing: BB1_16, BB1_17, BB2_18, BB3_02, BB3_05, CRISPR-PPP

• PCR: 41, 42, 45, 52, 53, 56, 57
Each PCR was loaded on a preparative gel and cut out before undergoing PCR-purification.

• Golden Gate Assembly: BB1_19, BB2_19, BB2_25, BB2_26, BB2_27, BB2_28, BB2_29, BB2_30, BB2_31, BB2_32, BB2_33, BB2_34, BB3_01 (unsuccessful), BB3_02, BB3_03 (unsuccessful), BB3_06, BB3_10, BB3_13-15, BB3_24
GG-products are then transformed into competent cells. Colonies with specific antibiotic resistances were selected out by using antibiotic-containing media. The next day, positives clones were confirmed via a OneTaq colony PCR and a gel electrophoresis before a Miniprep was made.

• Preculture of DH10B E. coli strain made chemically competent

• Yeast cells transformed with the Cas9 plasmid

• Sent for Sequencing: BB1_19, CRISPR 2, CRISPR 3, BB3_12, BB3_13, BB3_14, BB2_34

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