Team:UC Chile/Cyano/Labook/july

July

- The following digestions were done:

RS1 + Kanr + psB1C3
RS1 + Kanr
B0014 + RS2 + psB1C3
B0014 + RS2

There were colonies in all the plates. They were cultured.

- Did a PCR run of LuxCDEG. Gel from electrophoresis showed: nothing.
- Gel with LuxCDEG (cut wit X+P and uncut): nothing. Ligations must be incorrect.
- Prepared all the material to send to Fernán at Cambridge.

The following ligations were done.

(Lux CDEG1 + E + S) digestion + (Lux CDEG2 + X + P) digestion + (psB1C3 + E+ P) digestion.

Colonies did not grow on Kanamycin plates. Negative colony PCR.
New digestion: LuxCD, LuxEG + terminator.
Negative colony PCR for RS2 + B0014 + psB4K5.

We could not identify LuxCD and LuxEG from VB. Anyway, they were ligated and transformed.

Synechocystis transformation:

psB1C3_IntC and RFP
For the psbAB + GFP transformation the synechocystis transformation protocol was followed until plating.
Colony PCR for LuxCEDG, B0014 + RS2 + KanR and psB4K5 + sfGFP: new failed attempt. New strategy: purification of RS2 and B0014 and B0015.

As B0014 is problematic the part was switched for B0015. The part was digested.
Also digested RS2 with (E + P) and ligated with B0015. Transformation and plating followed.

Another ligation to assemble: (RS1 + Kan) (E + S) + (RS2)(X + P)... LuxCD + LuxEG to intC.
PCR for LuxCDEG plasmid.

Parts purified (RS1 + Kan) , RS2, B0014 and B0015.

Ligations: (RS1 + Kan) + RS2 in psB1A2
(RS2 + B0014) in psB1C3
(RS2 + B0015) in psB1C3
LuxCD + LuxEG in Int_C

New PCR for LuxCD and LuxEG with VF (suffix R_digest) and VR (prefix F_digest)
LuxCD looks faint, LuxEG is good. Band LuxEG was extracted.

Gel with colony PCR RS2 cut (no restriction site) and LuxCDEG.
The bands with normal size were ligated to B0014 and B0015.

PCR runs for psB1C3, psB1T3, psB1K3, psB1K5 and LuxCD.

Colony PCR for ligations (included RS1 + RS2) All had correct size except LUX CD + EG)
Finally digested LuxCD from Brick and Lux EG from PCR. LuxCD: size was correct, ligated back and transformed.
Minipreps for colony PCR. DNA was extracted, parts were digested and ligated.

RS1 + Kan + B0014 + RS2 in psB1C3
RS1 + Kan + B0015 + RS2 in psB1C3

Will be used to insert parts between RS1 and Kan by Gibson.

Different PCR's to obtain: VB_pBAD, RS1 + RS2, Lux CD, psB1A3, pSb1t3. Low yield (except VB_pBAD and RS1 and RS2). PCR was done with this parts as template to amplify back.

Colony PCR for Lux ligations, RS1 + Kan + B0014 + RS2 in psB1C3 and RS1 + Kan + B0015 + RS2 in psB1C3.
Right size: RS1 + Kan + B0014 + RS2 + psB1C3
RS2 + Kan + B0015 + RS2 + psB1C3

Lux CDEG will be miniprepped and size will be checked.

New PCR for parts: ADF-3, psB1C3, LuxAB

minipreps: LuxCD, LuxCDEG, RS1 Kan r + B0015 + RS2, RS1 + Kanr + B0015 +RS2, RS1 + Kanr + B0014 + RS2.

By verification digest (E + P):
LUXCDEG wrong size, B0015 wrong size, B0015 right size (colony 1).
C4 is ready!

So now we have 3 basic problems.
- Not sure if primers amplified our pieces (if done with preffix, suffix, no restriction enzyme will join).
- When digest CD and EG lots of pieces are loose. As ligase also nicks blunt ends, there are lots of false positives
- CD, EG and psB1C3 have the same size so they can't be told apart by electrophoresis. New ways to ligate LuxCDEG:

1. amplify CD and EG (VF2 and VR)
2. digest LuxCD (E+S), LuxEG (X+P)
3. Electrophoresis (now parts can be distinguished)
and/or
4. ligate digest LuxCD from plasmid (S+P)
5. ligate with LuxEG

PCR for: translado promoter, Pcaa3, LuxAB, VB (psB1C3, psB1K3, psB1A2, pSB1T3)
So, PCR and electrophoresis for LuxCD and EG (right size).
New batch of competent cells.
Ligation LuxCD + LuxEG + psB4K5
LuxCD + LuxEG + psB1K3

New Gibson attempt. sfGFP in psB4K5 (just a try out for new competent cells)
Transformation in pUC 19
PUC had colonies. Ligations with colonies. Positive control for Gibson (sfGFP in psB4K5) turn out to be positive :) So the problem was: E. cloni instead of TOP 10.
Colony PCR for LuxCDEG in psB4K5. Had red colonies in it.
PCR for RS1_Kanr_B0015_RS2
As primers form dimers the PCR was done at hgher Tm
amplification of RS1+ Kan + B001 + RS2 --> right size