Difference between revisions of "Team:British Columbia/Notebook"
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<p>First, combining F3H and B0034:</p> | <p>First, combining F3H and B0034:</p> | ||
<ul> | <ul> | ||
| − | <li> | + | <li> Transform from distribution kit wells via heat shock method</li> |
| − | <li> | + | <li> Spread on CMB-LB agar plate at 37c° overnight |
| − | <li> | + | <li> Pick and inoculate with LB+Cm 37c° for 16 hours </li> |
| − | <li> | + | <li> Miniprep to obtain plasmids (elution buffer, 50μm) </li> |
| − | <li>Spread on CMB-LB agar plate | + | <li> Digest F3H with Xba1</li> |
| + | <li> Spread on CMB-LB agar plate at 37c° overnight </li> | ||
<li>PCR and gel electrophoresis to analyze if successful </li> | <li>PCR and gel electrophoresis to analyze if successful </li> | ||
<li> Take positive colonies and place in LB with CMP incubate for 16 hours at 37*C </li> | <li> Take positive colonies and place in LB with CMP incubate for 16 hours at 37*C </li> | ||
Revision as of 01:10, 18 October 2018
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)
Biosensor Notebook
July
- 26:
- Transformed RFP + RBS into DH5a
- Transformed GFP + RBS into DH5a
- 27:
- Inoculated RFP + RBS in 25mg/uL of CM-25
- Inoculated GFP + RBS in 25mg/uL of CM-25
- 28:
- Miniprepped RFP + RBS
- Miniprepped GFP + RBS
- Digested RFP + RBS with ECOR1 and XBA1 to obtain 8.96uL of DNA at 111.16ng/uL
- Digested GFP + RBS with ECOR1 and XBA1 to obtain 12.8uL of DNA at 78.1ng/uL
- Eventually discarded both digests as we did not use them
August
- 7:
- Transformed Bba_E0020 into DH5a
- 8:
- Inoculated CFP strains with 25mg/uL of CM-25
- 9:
- Digested CFP with ECOR1 + XBA1 to obtain 8.77uL of DNA at 114.0ng/uL
- 15:
- Transformed RFP into DH5a
- 16:
- Inoculated RFP in 25mg/uL of CM-25
- Miniprepped and digested RFP with ECOR1 + XBA1 to obtain 9.14uL of DNA at 109.4ng/uL
- 18:
- Transformed Bba_E0040 (GFP) into DH5a
- Transformed Bba_l15016 (CFP + RBS) into DH5a
- Synthesized parts from IDT were resuspended in 1X TBE
- FdeR
- Pt181-repressor
- Pt181-GP2
- GP2
- Antisense
- RFP, RFP+RBS, CFP, GFP+RBS have all been digested
- 19:
- Transformed Bba_E0040 (GFP) into DH5a
- Transfroemd CFP + RBS into DH5a
- Both transformations were unsuccessful
- 20
- OD of DH5a competent cells was 0.69
- Pelleted competent cells using 0.1M CaCl2
- Transformed Bba_E0040 (GFP) into DH5a
- Transformed Bba_I5016 (CFP + RBS) into DH5a
- 21:
- OD of DH5a competent cells was 0.53
- Pelleted competent cells using 0.1M CaCl2
- Positive and Negative controls did not function properly
- Realized glycerol was not added
- Re-setup experiment
- 23:
- Reaction cleanup using ABM kit, involving:
- GP2
- E+S
- Pt181-GP2
- FdeR
- Transformed Bba_E0040 (GFP) into DH5a
- Transformed Bba_I15016 (CFP + RBS) into DH5a
- Both transformations were unsuccessful
- 24
- Ran plasmid PSB1C3 digested with X+P, with 1X TAE buffer
- Ran plasmid PSB1C3 digested with E+S, with 1X TAE buffer
- Ran plasmid PSB1C3 digested with E+P, with 1X TAE buffer
- Purified with ABM kit:
- X+P
- E+P
- E+S
- CFP
- GFP+RBS
- RFP+RBS
- RFP
- Inoculated CFP and GFP in 25mg/uL of CM-25
- 25:
- Miniprepped CFP+RBS and GFP (concentration 149ng/uL)
- 27:
- Ligated FdeR and Repressor DNA into RFP vector
- Ligated FdER and Repressor DNA into E+S vector
- Ligated pt181-GP2 and GP2 DNA into E+P
- Ligated antisense DNA into X+P
- 29:
- Transformed in DH5a: RFP - FdeR, RFP - Repressor, E+S - FdeR, E+S - Repressor, CFP - Repressor, CFP - FdeR, E+P - Pt181-GP2, E+P - GP2, X+P - Antisense
- 30:
- Inoculated in 25mg/uL of CM25: RFP - FdeR, RFP - Repressor, E+S - FdeR, E+S - Repressor, CFP - Repressor, CFP - FdeR, E+P - Pt181-GP2, E+P - GP2, X+P - Antisense
- 31:
- Miniprepped: RFP - FdeR, RFP - Repressor, E+S - FdeR, E+S - Repressor, CFP - Repressor, CFP - FdeR, E+P - Pt181-GP2, E+P - GP2, X+P - Antisense
Spetember
- Digested: RFP - FdeR, RFP - Repressor, E+S - FdeR, E+S - Repressor, CFP - Repressor, CFP - FdeR, E+P - Pt181-GP2, E+P - GP2, X+P - Antisense
- Ran Gel Electrophoresis In 1X TBE buffer on: RFP - FdeR, RFP - Repressor, E+S - FdeR, E+S - Repressor, CFP - Repressor, CFP - FdeR, E+P - Pt181-GP2, E+P - GP2, X+P - Antisense
- Double checked digestions with BamH1 + ECOR1 + Pst1 of strains: RFP - FdeR, E+S-FdeR
- Transformed RFP - FdeR2 into DHd5a
- Digested GFP Vector using: E+P, E+X, E+S
- Ran Gel Electrophoresis on: GFP Vector + (E+P, E+X, E+S)
- Purified: GFP - E+P, GFP - E+S
- Digested Repressor (from IDT) using E+S
- Ligated repressor with E+S vector, pt181 GP2 and GP2 with E+P digested vector. Antisense ligated with X+P vector
- Inoculated: E+S - Rep Lig 1, E+P - pt181GP2 Lig, E+P - GP2 Lig, Antisense - X+P Lig
- Miniprepped: E+S - Rep Lig 1, E+P - pt181GP2 Lig, E+P - GP2 Lig, Antisense - X+P Lig
- Digested with E+P: E+S - Rep Lig 1, E+P - pt181GP2 Lig, E+P - GP2 Lig, Antisense - X+P Lig
- Performed Gel Electrophoresis of DNA strains: E+S - Rep Lig 1, E+P - pt181GP2 Lig, E+P - GP2 Lig, Antisense - X+P Lig
Kaempferol 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)
F3H
BBa_K1497009
~1107 BP
Spring 2018 Distribution
Plate 2, Well 12I
pSB1C3 (Cm)
RBS
BBa_B0034
~12 BP
Spring 2018 Distribution
Plate 4, Well 1N
pSB1C3 (Cm)
Double Terminator
BBa_B0015
~129 BP
Spring 2018 Distribution
Plate 3, Well 3F
pSB1C3 (Cm)
First, combining F3H and B0034:
- Transform from distribution kit wells via heat shock method
- Spread on CMB-LB agar plate at 37c° overnight
- Pick and inoculate with LB+Cm 37c° for 16 hours
- Miniprep to obtain plasmids (elution buffer, 50μm)
- Digest F3H with Xba1
- 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
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)