• 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
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.

X

X

X

X

X

X

X

X

• 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 of 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

X

X

X

X

X

X

X

X

• 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, C-PPP_01 (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

X

X

X

X

X

X

X

X

X

X

X

X

X

• 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, BB1_18, BB3_02, BB3_05, C-PPP_01

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

• 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, BB3_14, BB3_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, BB2_34, BB3_12, BB3_13, BB3_14, C-PPP_02, C-PPP_03

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

• Golden Gate Assembly: BB2_36, BB3_07, BB3_10, BB3_11, BB3_13, BB3_14, BB3_17, BB3_18, BB3_19, BB3_20, BB3_21, BB3_22, BB3_23, BB3_24, C-PPP_03, C-PPP_04
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.

• A temperature-sensitivity test was performed for cells with pSIM5, a temperature sensitive plasmid. An overnight culture was incubated at 37°C and a control was incubated at 28°C. The overnight culture at 37°C was tested as positive and lost the plasmid, while the control maintained its plasmid.

• Two clones of yeast cells carrying the Cas9 plasmid made competent for the next transformation

• Sent for Sequencing: BB2_19, BB2_21, BB2_31, BB2_32, BB2_33´, BB2_34, BB3_01, BB3_13, BB3_14, C-PPP_02, C-PPP_03

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

• Golden Gate Assembly: BB1_26, BB2_23, BB3_02 (unsuccessful), BB3_12, BB3_16, BB3_17, BB3_20, BB3_22, BB3_28, C-PPP_05, C-PPP_06
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.

• Overnight cultures of C-PPP_04 tested for temperature sensitivity

• Sent for Sequencing: C-PPP_05, C-PPP_06, BB3_20, BB3_22

X

X

X

X

X

X

X

X

Everything we did in the eighth week...

Everything we did in the ninth week...

Everything we did in the tenth week...

Everything we did in the eleventh week...

Everything we did in the twelfth week...

Everything we did in the thirteenth week...

Everything we did in the fourteenth week...

Everything we did in the fifteenth week...