igem2016 Microbiology BMB SDU

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1 igem2016 Microbiology BMB SDU Project type: Plastic Project title: Additional promoter RBS biobricks Sub project: 1. K K & K K , K , K & K Creation date: Written by: Joel Mario Vej-Nielsen Performed by: The entire igem team 1. SOPs in use. SOP0001 SOP0005 Freeze stock SOP LA plates with antibiotics - (SOP is not mentioned in protocol.) SOP0009 TSB SOP0014 Gel purification SOP Ligation SOP0017 Fast Digest SOP0019 plasmid miniprep SOP0019 plasmid miniprep SOP Colony PCR with MyTag SOP Ca Purpose. To create a library of k with different additional promoter and RBS combinations. 3. Overview. Day SOPs Experiments 1 SOP0009 TSB- of K & K into E. coli. 2 SOP0021 SOP0001 Colony PCR on K & K containing cells. of k & K containing cells. 3 SOP0019 SOP0017 SOP0014 SOP0015 Plasmid MiniPrep of K & K Fast Digest of K & K Gel purification on K & K Ligation of K insert with K backbone (K ). Page 1 of 10

2 4 SOP0009 TSB- of K ligations into E. coli. 5 SOP0021 Colony PCR to identify successful ligation. 6 SOP0001 of bacteria with K SOP0005 Freeze stock of bacteria containing K SOP0009 TSB- of J23106 & K into E. coli. 9 SOP0001 of J23106 & K containing cells. 10 SOP0019 of J23106 & K SOP0017 Fast Digest of J23106 & K SOP0014 Gel purification of linearized J23106 & a K insert. 11 SOP0009 TSB- of J23104 into E. coli. 12 SOP0001 of J23104 containing cells. 13 SOP0019 of J23104 SOP SOP0014 SOP0015 Fast Digest of J Gel purification of linearized J Ligation of the K insert into the J32104 (K ) and the J23106 (K ) backbone. 15 SOP0023 Ca 2+ of K ligation into E. coli. 16 SOP0001 s of K containing cells. 17 SOP0019 Miniprep på K containing cells. 18 SOP0023 Transformation of K into E. coli 19 SOP0001 of K containing cells. 20 SOP0019 SOP0017 Miniprep på K containing cells. Fast Digest of K & K in order to check if the size is correct, as colony PCR was giving two bands. 21 SOP0023 Ca 2+ Transformation of J23104 & J23106 into E. coli. 22 SOP0001 ON Culture of K & K containing cells. 23 SOP0019 Miniprep on K & K SOP0017 Fast digest of K & K SOP0014 Ligation of K & K into psb1c3. 26 SOP0023 Ca 2+ of K & K ligations into E. coli. 27 SOP0001 s of K & K containing cells. 28 SOP0005 Freeze stock of bacteria containing K & K SOP0009 TSB- of K608003, K608004, K & K into E. coli. 30 SOP0001 of K608003, K608004, K & K containing cells. 31 SOP0019 of K608003, K608004, K & K SOP0017 Fast Digest of K608003, K608004, K & K SOP0014 SOP0015 Gel purification of linearized K608003, K608004, K880005, K & a K insert. Ligation of the K into the K backbone (K ), the K (K ), the K (K ) & the K backbone (K ). Page 2 of 10

3 34 SOP0023 Ca 2+ of K , K , K & K ligations into E. coli. 35 SOP0001 s of K , K , K & K containing cells. 36 SOP0005 Freeze stock of bacteria containing K , K , K & K SOP0005 Freeze stock of bacteria containing K & K Page 3 of 10

4 4. Materials required. Materials in use Name Components (Concentrations) Manufacture r / Cat. # Room Safety considerations LB Sigma? Chem room LA Sigma? Chem room Chloramphenicol 30mg/ml yes Ampicillin 100mg/ml Miniprep plasmid kit Thermo Fischer igem work station Gel purification kit Thermo Fischer igem work station Fasdigest enzymes: Agilent igem freezer EcoRI, XbaI, SpeI & PstI Technologies Ligase igem freezer Fast digest buffer Agilent Technologies igem freezer Ligase buffer Glycerol 50% Agilent Technologies igem freezer Taq DNA polymerase Ampliqon igem freezer Polyethylene glycol 3,350 Sigma Aldrich Chem room Dimethyl sulfoxide Sigma Aldrich Fuming hood Magnesium Chloride 1M Page 4 of 10

5 5. Other 6. Experiment history. Date SOPs (YY.MM.DD) SOP0009 TSB SOP0021 Colony PCR Alterations to SOPs and remarks to experiments Every biobrick in this SOP was worked with here, but only the work with K was finished. Deviations: - PCR settings Step 1 Initial duration SOP SOP SOP0017 Fast digest SOP0014 For K Step 2 Denaturation 95 Step 3 Annealing 60 Step 4 Extension/Elongation 72 Step 5 Repeat of step 2-4 Step 6 Extra Elongation 72 Step 7 Keep the samples cold 20 - Another polymerase Tag2x Mastermix RED AMPLIQON - Another dilutions of master mix: For one PCR sample: Primers: VF2 and VR 1 µl of each Tag 2x: 5µL Water: 2.5 µl Template sample: 0.5µL For K After eluting DNA, solution was added to filter a second time, incubated for two minutes at room temperature and spun for two minutes at rpm. For K Due to poor concentration the amount of buffer and the final volume of the reaction was doubled for PR4. K was digested with XbaI and PstI K was digested with SpeI and PstI Page 5 of 10

6 Gel purification SOP0015 T4 ligation SOP0009 TSB SOP0021 Colony PCR The amount of substance wasn t calculated, instead the two samples were added in a volume K608002:k (k201800) of 1:0, 1:2 & 1:5. K into E. coli. Deviations: - PCR settings Step 1 Initial duration SOP SOP0005 Freeze stock SOP0009 TSB Step 2 Denaturation 95 Step 3 Annealing 60 Step 4 Extension/Elongation 72 Step 5 Repeat of step 2-4 Step 6 Extra Elongation 72 Step 7 Keep the samples cold 20 - Another polymerase Tag2x Mastermix RED AMPLIQON - Another dilutions of master mix: For one PCR sample: Primers: VF2 and VR 1 µl of each Tag 2x: 5µL Water: 2.5 µl Template sample: 0.5µL For K SOP SOP0019 For J SOP0017 For J23106 with SpeI and PstI After the storage of this first biobrick, the other were created, but due to a mutation in K and poor storage of DNA we had to start over with new bricks from the igem kit. DNA fresh from the igem kit. Again work was done with all the remaing bricks but failed for all but J23104 (K ) & J23106 (K ). For J23106 Page 6 of 10

7 Fast Digest SOP0014 Gel purification SOP0009 TSB SOP SOP SOP0017 Fast digest SOP0014 Gel purification SOP0015 T4 ligation SOP0023 Ca SOP SOP SOP0023 Ca SOP SOP SOP0017 Fast digest SOP0014 Gel purification SOP0015 T4 ligation SOP0009 TSB er Work was halted for the linearized J23106 until the rest of the DNA was ready to be ligated as well. Of J23104 into E. coli. of J23104 containing cells of J23104 Fast digest of J23104 with SpeI and PstI Purification of linearized J23104 The amount of substance was not calculated, instead the two samples were added in a volume J23104:K (K ) J23106:K (K ) of 1:0, 1:2 & 1:5. Transformation of K and K into E. coli. No growth on K containing cells. of K containing cells. Miniprep of K This DNA was stored while the rest of the biobricks were assembled Transformation of K into E. coli. of K containing cells of K Digestion of K & K with SpeI and PstI Of linearized K & K Ligation of K & K into psb1c3. Ligated 1:1. Transformation of K & K into E. coli. This time they are in psb1c3. Page 7 of 10

8 SOP0001 of cells containing K & K SOP0005 Freeze stock is made of K (J23104) & K (J23106). Freeze stock SOP0009 K608003, K K & K from the igem kit. TSB SOP0001 Of cells containing K608003, K K & K SOP0019 Plasmid purification of K608003, K K & K SOP0017 Digests of K608003, K K & K Fast digest With spei and PstI SOP0014 Gel purification of linearized K608003, K K & Gel purification K SOP0015 The amount of substance wasn t calculated, instead the two T4 ligation samples were added in a volume K608003:k (K ) K608004:K (K ) K081005:K (k ) k880005:k (k ) SOP0009 TSB SOP SOP SOP SOP0005 Freeze stock of 1:0, 1:2 & 1:5. Transformation of K , K , K & K Ligation of K , K , K & K Plasmid purification of K , K , K & K of cells containing K , K , K & K Freeze stock of cells containing K , K , K & K Sample specification. Page 8 of 10

9 Sample name Sample content From Used for / Saved where PR4 BBa_K kit, plate 2, Fast digest well 11C PR5 BBa_K kit, plate 1, Fast digest well 3O PG7 BBa_K Cut with XbaI PR4 Ligation & PstI PG3 BBa_K cut with SpeI PR5 Ligation & Pst. PR10 BBa_k PG3 & PG7 Saved as #8 PR26 BBa_J kit, plate 4 Fast digest well 17L PG18 BBa_J23104 PR26 Ligation PR3 BBa_J kit, plate 4, Fast digest well 17P PR22 BBa_K From PR4 Fast digest PG26 BBa_J23106 cut with SpeI & PR3 Ligation PstI PG27 BBa_K PR22 Ligation PR42 BBa_K PG18 & PG27 Fast digest PR43 BBa_K PG26 & PG27 Fast digest PG31 BBa_K PR42 Ligation PG32 BBa_K PR43 Ligation PR42 BBA_K PG31 & PG33 Saved as #64 PR43 BBa_K PG32 & PG33 Saved as #65 PR59 BBa_K kit, plate 2 Fast digest well 3F PR60 BBa_K kit, plate 3 Fast Digest well 15F PR61 BBa_K kit, plate 1 Fast digest well 5A PR62 BBa_K kit, plate 1 Fast digest well 5C PG48 BBa_K PR59 Ligation PG49 BBa_K PR60 Ligation PG50 BBa_K PR61 Ligation PG51 BBa_K PR62 Ligation PG53 BBa_K PR65 Ligation PR69 BBa_K PG48 & PG53 Stored as #91 PR70 BBa_K PG49 & PG53 Stored as #92 PR71 BBa_K PG51 & PG53 Stored as #94 PR72 BBa_K PG50 & PG53 Stored as #93 Page 9 of 10

10 8. Remarks on setup. When K and K K the remaining biobricks were also attempted to be synthesized. However, it failed and therefore the complete synthesize took three rounds. 9. Results and conclusions. A library of BioBricks that can produce PHB. 10. Appendixes Page 10 of 10