• Preparation of 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 an 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 an 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 of 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 an 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 of 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 an 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 of 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 an 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 of 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 an 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

• Golden Gate Assembly of 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 an 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

• PCR: DH5-α pSIM5, #71, #72
Each PCR was loaded on a preparative gel and cut out before undergoing PCR-purification according to protocol.

• Golden Gate Assembly of BB1_27, BB3_01, BB3_03, BB3_30, BB3_31, BB3_32, BB3_33
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 an OneTaq colony PCR and a gel electrophoresis before a Miniprep was made.

• BB3_32 cultivated in YP-G418, a yeast peptone media

• Transformation of pSIM5 and BB3_31 into yeast cells

X

X

X

X

X

X

X

Improving parts: For more information please visit: Improve

•PCR: BB3_35, pSIM5
Each PCR was loaded on a preparative gel and cut out before undergoing PCR-purification according to protocol. https://static.igem.org/mediawiki/2017/0/06/T--BOKU-Vienna--BB335.png

•Golden Gate Assembly of BB3_1, C-PPP_08, C-PPP_09, C-PPP_10, BB3_33, C-PPP_07, BB3_33
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 an OneTaq colony PCR and a gel electrophoresis before a Miniprep was made.

•Minipreparation for plasmid DNA extraction: BB3_12 + 30, BB3_15

•Transformation: pSIM9

X

• PCR: #88, #89, #90
Each PCR was loaded on a preparative gel and cut out before undergoing PCR-purification

• Golden Gate Assembly of BB3_12, BB3_34, BB3_35 (special), BB3_35, BB3_36, BB3_37, BB3_38, C-PPP_12
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 an OneTaq colony PCR and a gel electrophoresis before a Miniprep was made.

• Lethality-Test of C-PPP_11 and C-PPP_12

X

X

X

X

X

• Golden Gate Assembly of BB3_27, BB3_36, BB3_37, BB3_38, BB3_39, BB3_40, BB3_43, C-PPP_10, C-PPP_11, C-PPP_12, C-PPP_12 (+ #97)
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.

• Transformation and OneTaq colony PCR of yeast

X

X

X

X

X

X

X

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...
•