Rehydration of Registry DNA

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

1. Add 10 μL of ddH₂O to the desired well.
2. Pipette up and down 3-5 times.
3. Incubate at room temperature for 10 minutes.
4. Transform cells with 1 μL of rehydrated DNA. Store the remaining amount at -20°C.

Rehydration of IDT Synthesized DNA

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

1. Centrifuge tube containing the synthesized DNA for 3-5 seconds at 3000g to ensure that all material is at the bottom of the tube.
2. Add ddH₂O to reach a final concentration of 50 ng/μL.
3. Vortex.
4. Incubate at 50°C for 20 minutes.
5. Briefly vortex and centrifuge . Store at -80°C.

Plasmid MiniPrep from Escherichia coli

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):

• 50 mM Tris-HCl, pH 8

• 10 mM EDTA

• 100 μg/mL RNase A

Lysis buffer:

• 200 mM NaOH

• 1% (v/v) SDS

Precipitation buffer:

• 3 M CH₃CO₂K, pH 5.5

Isopropanol

70% ethanol

Table-top centrifuge

Ice bucket

2-mL microcentrifuge tubes

1.5-mL microcentrifuge tubes

ddH₂O 3

Protocol

1. Transfer 2 mL of the overnight culture to a 2-mL microcentrifuge tube and pellet the cells by spinning at 3500 g for 1 minute. Discard supernatant.
2. Resuspend pellet in 300 μL Resuspension buffer.
3. Add 300 μL Lysis buffer. Invert gently and incubate at room temperature for 3-5 minutes.
4. Add 300 μL Precipitation buffer. Invert gently. A white precipitate should form.
5. Centrifuge at 14,000g for 10 minutes at room temperature.
6. Retain supernatant in a clean 1.5-mL microcentrifuge tube.
7. Add 650 μL isopropanol. Gently invert and incubate at room temperature for 10 minutes.
8. Centrifuge at 14,000g for 10 minutes at 4°C. Discard supernatant.
9. Wash pellet with 500 μL cold 70% ethanol. Add to microcentrifuge tube. Do not resuspend.
10. Centrifuge at 14,000g for 5 minutes at 4°C. Discard supernatant.
11. Dry pellet in speed vac for 15-30 minutes, or until no more liquid remains visible in the tube.
12. Resuspend pellet in ddH₂O and store at - 20°C.

Restriction Digest

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

1. Into a 0.2-mL PCR tube add the following:

• 1 μg DNA

• 1 μL restriction enzyme 1

• 1 μL restriction enzyme 2

• 2 μL 10X appropriate buffer

• 1 μL 100X BSA, if using

• ddH₂O to a final volume of 20 μL

2. Incubate at 37°C for one hour.
3. Deactivate restriction enzymes via heat shock by incubating tube at 80°C for 20 minutes.

Ligation of DNA Inserts to Plasmid Backbones

Experimental Details and Rationale

Insert text

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

1. To a 0.2-mL PCR tube add:

• 50 ng digested vector DNA

• Appropriate amount of digested insert DNA to give a 3:1 molar ratio of insert:vector

• 1 μL T4 DNA ligase

• 2 μL 10X T4 DNA ligase buffer

• ddH2O to a total volume of 20 μL

2. Incubate tube at room temperature overnight.
3. Use 10 μL to transform cells, store at -20°C.