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Revision as of 22:12, 20 July 2017
Our Experiments
General Protocols
Experimental Details and Rationale |
Registry DNA was rehydrated for completion of the Interlab Study. Also, Part BBa_K934001 (phaC1-A-B1) was rehydrated by the Secretion subgroup and transformed into our chassis so that P(3HB) was produced and preliminary secretion assays could be performed before the Synthesis subgroup had completed their cloning. |
Materials |
iGEM 2017 distribution kit ddH₂O |
Protocol |
|
Experimental Details and Rationale |
Our genetic parts were ordered from IDT and arrived as a dry, lyophilized powder. They were resuspended in aqueous solution for cloning into pSB1c3 or pET29B vectors and to ligate multiple parts together. |
Materials |
Synthesized DNA from IDT (gBlocks) ddH₂O |
Protocol |
|
Experimental Details and Rationale |
Antibiotics were added to agar to select for successful E.coli transformants. The vector pSB1c3 was selected for with chloramphenicol and pET29B was selected for with kanamycin. |
Materials |
Luria-Bertani broth with agar:
Appropriate antibiotic:
dH2O 1500-mL Erlenmeyer flask Stir bar Aluminum foil |
Protocol |
|
Experimental Details and Rationale |
Culture broth was plated on agar to isolate single colonies of E.coli. |
Materials |
Luria-Bertani agar plate with appropriate antibiotic (if required) Overnight culture of desired bacteria 70% ethanol Spreading rod Bunsen burner |
Protocol |
|
Experimental Details and Rationale |
Culture broth was streaked on agar to isolate single colonies of E.coli. |
Materials |
Luria-Bertani agar plate with appropriate antibiotic (if required) Overnight culture of desired bacteria or single isolated colony on agar plate Inoculation loop Bunsen burner |
Protocol |
|
Experimental Details and Rationale |
E. coli DH5ɑ and BL21 were lysed and the pSB1c3 or pET29B vectors were isolated to be used in cloning our genetic constructs. Bacterial clones were lysed for analysis (eg: confirmation restriction digest, genetic sequencing). |
Materials |
2.5 mL overnight culture of bacteria in Luria-Bertani broth with appropriate buffer in 16x125mm culture tube Resuspension buffer (stored at 4°C):
Lysis buffer:
Precipitation buffer:
Isopropanol 70% ethanol Table-top centrifuge Ice bucket 2-mL microcentrifuge tubes 1.5-mL microcentrifuge tubes ddH₂O 3 |
Protocol |
|
Experimental Details and Rationale |
Our genetic parts and vectors were digested with restriction enzymes before they were ligated. Plasmids isolated from transformants (through Plasmid Miniprep) were also digested for confirmation of ligation and transformation. |
Materials |
DNA (eg: from plasmid miniprep) Restriction enzymes 10X appropriate buffer ddH₂O 100X Bovine Serum Albumin (BSA) (if using PstI) 0.2-mL PCR tubes |
Protocol |
|
Experimental Details and Rationale |
Insert text |
Materials |
Insert material 1 Insert material 2 insert material 3 etc. |
Protocol |
|
Experimental Details and Rationale |
Digested registry DNA or digested genetic parts from IDT were ligated to either pSB1c3 or pET29B for propagation in E.coli DH5ɑ or protein expression in E.coli BL21. Later, our parts were ligated to pSB1c3 for submission to the iGEM registry. |
Materials |
Digested vector DNA Digested insert DNA 10X DNA ligase buffer (from New England BIolabs) T4 DNA ligase (1 U/ μL) (from New England Biolabs) ddH2O 0.2-mL PCR tubes |
Protocol |
|
Experimental Details and Rationale |
Insert text |
Materials |
Insert material 1 Insert material 2 insert material 3 etc. |
Protocol |
|
Experimental Details and Rationale |
Insert text |
Materials |
Insert material 1 Insert material 2 insert material 3 etc. |
Protocol |
|
Experimental Details and Rationale |
Insert text |
Materials |
Insert material 1 Insert material 2 insert material 3 etc. |
Protocol |
|
References
Rose, C., Parker, A., Jefferson, B., & Cartmell, E. (2015). The Characterization of Feces and Urine: A Review of the Literature to Inform Advanced Treatment Technology. Critical Reviews In Environmental Science And Technology, 45(17), 1827-1879. http://dx.doi.org/10.1080/10643389.2014.1000761