Team:HokkaidoU Japan/Notebook/plastic Week 5

Transformation

We transformed pGEM into E.coli (strain:BL21). This transformation is the bioplastic production test in BL21.

1. Added 1 ul of plasmid DNA solution to 50 ul of thawed competent cells on ice.
2. Incubated on ice for 30 min.
3. Added 600 ul of LB.
4. Prepared and Labeled two petri dishes with LBA.
5. Plate 300 ul of the transformation onto LBA dish and spread.
6. Added 900 ul of LB to 100 ul of the transformation and plated 300 ul of it onto LBA dish then spread.
7. Incubated the plates at 37C for 15 hrs 30 min.

Liquid culture

Liquid culture of transformed BL21(DE3)

1. Added 2 ml of LBA into culture tubes.
2. Resuspended 2 colonies.
3. Incubated the tubes at 37C for 18 hrs.

After incubating, we tried to product PHB in BL21(DE3)

PHB production test

We prepared those three medium tubes and incubated them for 48 hrs.

1. 700 ml LB + 0.7 ul Amp
2. 700 ml LB + 0.7 ul Amp + 28 ul 50% glucose
3. 700 ml LB + 0.7 ul Amp + 28 ul 50% glucose + 1.4 ul Sodium pantothenate

• BL21(DE3) = No. 1~3 incubated
• JM109 = No. 2 only incubated (control)

Today the requested parts to HQ were sent! Parts were sent as agar stabs. We note here how to use these agar stabs. Using agar stab(http://partsregistry.org/Help:Requesting_Parts) We send out our part requests as agar stabs. The shelf life of these are short, so it is usually best to plate from the stab as soon as possible.
The agar should already have a hole from when it was stabbed. With an inoculating loop dipped into the stab, you can plate directly onto a petri dish with the appropriate antibiotic.

1. Incubate the dish overnight at 37C (14-16 hrs)
2. Pick a single colony to start up a culture
3. Extract plasmid DNA
4. Use the part!