Team:British Columbia/Notebook
Naringenin Notebook
We kindly received the following from UBC iGEM distribution kit of 2018:
Constitutive promoter + RBS
BBa_K880005
~70 BP
Spring 2018 Distribution
Plate 2, Well 3F
pSB1C3 (Cm)
BBa_K880005
~70 BP
Spring 2018 Distribution
Plate 2, Well 3F
pSB1C3 (Cm)
4CL
BBa_K801093
~1700 BP
Spring 2018 Distribution
Plate 2, Well 17D
pSB1C3 (Cm)
RBS+TAL
BBa_K1033000
~1600
Spring 2018 Distribution
Plate 4, Well 6C
pSB1C3 (Cm)
First, TAL and 4CL with RBS (ribosomal binding site) to combine them:
- 4CL digest with EcoRI + SpeI
- TAL digest with EcoRI + Xbal
- Ligation of both digestion with T4 ligase
- E. coli transformation via heat shock method
- Spread on CMB-LB agar plate and incubate at 37 C° overnight
- PCR and gel electrophoresis to analyze if successful
- Take positive colonies and place in LB with CMP incubate for 16 hours at 37*C
- Plasmid obtained
Second, we will take bio bricks of CHS + CHI with RBS (ribosomal binding site):
- CHS digest with EcoRI and SpeI
- CHI digest with EcoRI and Xbal
- Ligation of both digestion with T4 ligase
- E. coli transformation via heat shock method
- Spread on CMB-LB agar plate at 37c° overnight
- PCR and gel electrophoresis to analyze if successful
- Take positive colonies and place in LB with CMP incubate for 16 hours at 37 C°
- Plasmid obtained
Third, combine the two plasmids previously prepared:
- 4CL – TAL digest by EcoRI and SpeI
- CHS-CHI with EcoRI and Xbal
- Dephosphorylate CHS-CHI
- Ligate the two together with T4 ligation
- Add GFP via digestion and then ligation*
- E. coli transformation via heat shock method
- PCR and gel electrophoresis to analyze if successful
- Take positive colonies and place in LB and CMP incubate for 16 hours at 37 C°
Protocol for cloning promoter-RBS-4CL in pSB1C3
A. Restriction enzyme digest 1. Prepare the master mix in a microfuge tube by combining the following: Component Volume to add (ul) 10X NEB Buffer 2.1 6 NEB PstI restriction enzyme 4 Autoclaved distilled water 26 TOTAL 36 2. Zip-spin the master mix to collect all liquid at the bottom of the tube 3. With a pipette set to 25 ul, pipette each reaction up and down several times to mix 4. Vortex the master mix thoroughly 5. Zip-spin the master mix again 6. Aliquot the master mix into 3 aliquots of 14 ul (discard the remaining master mix) 7. Label the three tubes containing the aliquots as follows: a. P-RBS b. 4CL c. PDC (positive digest control)