Miniprep

- miniprep twice-overnighted cultures of P1010-Kan and P1010-Cm

- miniprep K145303, J23102, E0422 from the cultures in the fridge.  J23102 is looking a little odd...

- and get some of the plasmid sample of P1010-Amp and K098988 if we have any

- miniprep overnight growth of C0051//λcI protein, B0015//stop, B0034//RBS, and J23066//ribokey+stop

- run 1% agarose gels with a ladder and these parts to determine if the plasmids are there

Miniprepped: Qiagen Miniprep Protocol

P1010 Kan A (lost by accident, broth culture is still fine)

P1010 Kan B

P1010 Cm C

P1010 Cm D

P1010 Kan E from August 17th's sub-broth culture

P1010 Cm F from August 17th's sub-broth culture

K145303 Quad

J23102 RFP

E0422 ECFP

We are thinking that J23102 (const. promoter) and  E0422 (RBS+ECFP+stop) would make an ideal first system as they would produce a complete system with a visible effect if they worked.  Plasmid for both parts is in the -80 freezer. R0010 (pLac), B0034 (standard RBS), J06504 (mCherry CDS), B0015 (double transcription stop) would also make a good two stage system from parts we definitely have.

1.35 pm

Started Agarose Gel Electrophoresis Protocol

gelAugust19.tif

Once again all of the P1010 Kan failed.

Assembly

(*cough cough* right, I keep forgetting that the ribokey DOESN'T NEED an RBS... Sorry :S)

- recall that for assemblies with really short parts, we'd always planned to do traditional assembly, yes? B0015 and B0034 are short parts

- take the minipreps of C0051, B0015, B0034, and J23066; cut with restriction enzymes as follows

- purify the parts - this can be done by miniprep, gel, and purification from gel

  - FIND SOMETHING OTHER THAN ETHIDIUM BROMIDE to visualise - we need these fragments viable

- combine and ligate C0051 to B0015, and B0034 to J23066

- transform DH5α with these plasmids as per normal - C0051-B0015 is on an Amp/Kan plasmid, B0034-J23066 is on an Amp plasmid

Corrected Revised Assembly

- six possible assemblies:

  - A = R0010+J23066 --> pLac+ribokey, second stage assembly with K145303 ribolocked GFP for lac/IPTG-inducible GFP

  - B = J23102+J23066 --> constitutive promoter+ribokey, second stage assembly with K145303 ribolocked GFP for constitutive GFP (control purposes)

  - C = J23102+E0422 --> constitutive promoter+ECFP, will immediately produce constitutive ECFP

  - D = C0051+B0015 --> waiting for I0500+J23032 pAra+ribolock

  - E = R0010+B0034 --> pLac+RBS

  - F = J06504+B0015 --> mCherry+stop, combine with E in second stage assembly

Selecting assemblies A, B, C to perform

- need R0010, J23066, J23102, E0422, P1010-Cm (for ease sake)

Using the three-antibiotic assembly protocol from Gingko Bioworks (referenced on the Lab protocols page)

In for 15 min of 37^oC at 3:25 pm

...Gingko calls for 15min in the bath... openwetware calls for two hours... We'll go with the Gingko instructions for today, because they're our source for enzymes. If things fail, or have a far lower efficiency than we'd wanted, we'll see about longer incubation times at 37^oC.

In for 20 min of 80^oC at 3:40 pm

When it comes out of the 80^oC water bath, we're saving at least 20µL of all five to run a gel tomorrow for an after-the-fact test.

10 min of room temp incubation with T4 ligase at 4:15 pm

20 min of 80^oC at 4.30 pm

Transformed all three as per Transformation Protocol and plated 100 uL and 200 uL of each on LB+cm for overnight incubation

Cultures

overnight pick&culture at room temp of DH5α (for start a new batch of competent cells)

overnight pick&culture at 37^oC with shaking of K098988 and K081005

Plating

- If the ordered parts show up, streak culture them immediately - P1010s on the appropriate plate, I0500 on Kan, R0051 on Amp

Cryopreserved

P1010 Kan B

P1010 Cm C(D)

K145303

J23102

E0422

Started overnight room temp culture of DH5alpha to do a new batch of competent cells tomorrow.

started overnight cultures of K098988 and K081005.