"
Lab Aug 19 2009
From 2009.igem.org
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
1 Kb Ladder P1010 Kan B P1010 Cm C P1010 Cm D P1010 Kan E P1010 Cm F J23102 Const. Prom. E0422 ECFP
Visible No band Band Band No band Band (streaky) Band Band
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
plasmid restriction enzymes band/fragment to save C0051 EcoRI and SpeI (E,S) short/small/travel more B0015 EcoRI and XbaI (E,X) long/large/travel less B0034 SpeI and PstI (S,P) long/large/travel less J23066 XbaI and PstI (X,P) short/small/travel more
- 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
plasmid restriction enzymes
C0051 λcI repressor EcoRI and SpeI (E,S)
B0034 standard RBS XbaI and PstI (X,P)
J06504 mCherry EcoRI and SpeI (E,S)
B0015 transcription stop XbaI and PstI (X,P)
R0010 lactose promoter EcoRI and SpeI (E,S)
J23102 constitutive promoter EcoRI and SpeI (E,S)
J23066 ribokey+terminator XbaI and PstI (X,P)
E0422 RBS+ECFP+stop XbaI and PstI (X,P)
- 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 37oC 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 37oC.
In for 20 min of 80oC at 3:40 pm
When it comes out of the 80oC 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 80oC 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 37oC 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.
