Notebook.
All protocols we used for the methods can be found here:
Protocol
For further information regarding our Golden Gate Assembly products, please refer to our part collection site.
Week 1 (03/07-07/07)
• Preparation of growth media
• Interlab Challenge 2017: Transformations of the plasmids into E. coli DH5α
for more information, please visit our Interlab site.
• E. Coli DH10B cells made chemically competent
• Miniprep of GG plasmids:
BB1_01, BB1_02, BB1_03, BB1_04, BB1_05, BB1_06, BB1_07, BB1_08
Week 2 (10/07-14/07)
• 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
Week 3 (17/07-21/07)
• 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
Week 4 (24/07-28/07)
• 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
Week 5 (31/07-04/08)
• 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
Week 6 (07/08-11/08)
• 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
Week 7 (14/08-18/08)
• Golden Gate Assembly of BB1_26, BB2_23, BB3_02 (unsuccessful), BB3_12, BB3_16, BB3_17, BB3_20, BB3_22, BB3_28 (unsuccessful), 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
Week 8 (21/08-25/08)
• 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 (unsuccessful), 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.
• Transformation of BB3_32 and BB3_31 into yeast cells
• BB3_31 and BB3_32 cultivated in YP+G418, a yeast peptone media for testing of the GFP expression in a plate reader.
Week 9 (28/08-01/09)
Improving parts: PCRs for removing the illigal BsaI restricion site. Golden Gate Assembly of the PCRs and cloning into the pSB1C3. 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.
•Golden Gate Assembly of BB3_01 (unsuccessful), C-PPP_08, C-PPP_09, C-PPP_10, BB3_33, C-PPP_07
GG-products are then transformed into competent cells. Colonies with specific antibiotic resistances were selected out by using antibiotic-containing media. The next day, positive clones were confirmed via an OneTaq colony PCR and a gel electrophoresis before a Miniprep was made.
•Minipreparation for plasmid DNA extraction: BB3_12, BB3_30, BB3_15
•Transformation: pSIM9
Week 10 (04/09-08/09)
• 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
Week 11 (11/09-15/09)
• 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
Week 12 (18/09-22/09)
• PCR: #102, #103, #105, #106
Each PCR was loaded on a preparative gel and cut out before undergoing PCR-purification
• Golden Gate Assembly of BB2_56, BB3_09, BB3_42, BB3_44, BB2_56, C-PPP_26, C-PPP_27 (unsuccessful), C-PPP_15 (unsuccessful), C-PPP_31, C-PPP_34, C-PPP_28 (successful)
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 S288C made electro-competent
• Toulouse Collaboration: for further information, please visit our Collaboration site.
Week 13 (25/09-29/09)
• PCR: #5, #110, #126, #127, #131
Each PCR was loaded on a preparative gel and cut out before undergoing PCR-purification.
• Golden Gate Assembly of BB1_32, BB2_51, BB2_52, BB2_53, BB2_54, BB2_55, BB2_57, BB2_60, BB3_41 (unsuccessful), BB3_43, BB3_45, BB3_46, BB3_47, BB3_48, BB3_49, C-PPP_14, C-PPP_15, C-PPP_16, C-PPP_19, C-PPP_19, C-PPP_20, C-PPP_21, C-PPP_22 , C-PPP_28, C-PPP_31, C-PPP_32, C-PPP_33, C-PPP_34
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.
• Heidelberg Collaboration: for further information, please visit our Collaboration site
Week 14 (02/10-06/10)
• Golden Gate Assembly of C-PPP_37, C-PPP_38, C-PPP_39
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.
Week 15 (09/10-13/10)
• Lethality Test of C-PPP_38, C-PPP_39
• Transformation of C-PPP_38, C-PPP_39 into E.coli with pSIM9 → made electro competent and induced with lambda-red
• Integration: C-PPP_39 and GFPuv
