Team:UC Chile/Cyano/Labook/july

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<p><font size= "5">July</font></p>
<p><font size= "5">July</font></p>
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July:
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- The following digestions were done:
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RS1 + Kanr + psB1C3
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RS1 + Kanr
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B0014 + RS2 + psB1C3
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B0014 + RS2
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There were colonies in all the plates. They were cultured.
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- Did a PCR run of LuxCDEG. Gel from electrophoresis showed: nothing.
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- Gel with LuxCDEG (cut wit X+P and uncut): nothing. Ligations must be incorrect.
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- Prepared all the material to send to Fernan at Cambridge.
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The following ligations were done.
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(Lux CDEG1 + E + S) digestion + (Lux CDEG2 + X + P) digestion + (psB1C3 + E+ P) digestion.
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Colonies did not grow on Kanamycin plates. Negative colony PCR .
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New digestion: LuxCD,  LuxEG + terminator
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Negative colony PCR for RS2 + B0014 + psB4K5.
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We could not identify LuxCD and LuxEG from VB. Anyway, they were ligated and transformed.
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Synechocystis transformation:
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psB1C3_IntC and RFP
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For the psbAB + GFP transformation the synechocystis transformation protocol was followed until plating.
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Colony PCR for LuxCEDG, B0014 + RS2 + KanR and psB4K5 + sfGFP: new failed attempt. News strategy: purification of RS2 and B0014 and B0015.
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As B0014 is problematic the part was switched for B0015. The part was digested.
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Also digested RS2 with (E + P) and ligated with B0015. Transformation and plating followed.
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Another ligation to assemble: (RS1 + Kan) (E + S) + (RS2)(X + P)... LuxCD + LuxEG to intC.
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PCR for LuxCDEG plasmid.
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Parts purified (RS1 + Kan) , RS2, B0014 and B0015.
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Ligations: (RS1 + Kan) + RS2 in psB1A2
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                    (RS2 + B0014) in psB1C3
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                      (RS2 + B0015) in psB1C3
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    LuxCD + LuxEG in Int_C
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New PCR for LuxCD and LuxEG with VF (suffix R_digest) and VR (preffix F_digest)
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LuxCD looks faint, LuxEG is good. Band LuxEG was extracted.
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Gel with colony PCR RS2 cut (no restriction site) and LuxCDEG.
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The bands with normal size were ligated to B0014 and B0015.
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PCR runs for psB1C3, psB1T3, psB1K3, psB1K5 and LuxCD.
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Colony PCR for ligations (included RS1 + RS2) All had correct size except LUX CD + EG)
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Finally digested LuxCD from Brick and Lux EG from PCR. LuxCD: size was correct, ligated back and transformed.
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Minipreps for colony PCR. DNA was extracted, parts were digested and ligated.
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RS1 + Kan + B0014 + RS2 in psB1C3
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RS1 + Kan + B0015 + RS2 in psB1C3
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Will be used to insert parts between RS1 and Kan by Gibson.
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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.
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Colony PCR for Lux ligations, RS1 + Kan + B0014 + RS2 in psB1C3 and RS1 + Kan + B0015 + RS2 in psB1C3.
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Right size: RS1 + Kan + B0014 + RS2 + psB1C3
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                    RS2 + Kan + B0015 + RS2 + psB1C3
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Lux CDEG will be miniprepped and size will be checked.
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New PCR for parts: ADF-3, psB1C3, LuxAB
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minipreps: LuxCD, LuxCDEG, RS1 Kan r + B0015 + RS2, RS1 + Kanr + B0015 +RS2, RS1 + Kanr + B0014 + RS2.
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By verification digest (E + P):
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LUXCDEG wrong size, B0015 wrong size, B0015 right size (colony 1).
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C4 is ready1
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So now we have 3 basic problems.
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- Not sure if primers amplified our pieces (if done with preffix, suffix, no restriction enzyme will join).
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- When digest CD and EG lots of pieces are loose. As ligase also nicks blunt ends, there are lots of false positives
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- CD, EG and psB1C3 have the same size so they can't be told apart by electrophoresis. New ways to ligate LuxCDEG:
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1. amplify CD and EG (VF2 and VR)
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2. digest LuxCD (E+S), LuxEG (X+P)
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3. Electrophoresis (now parts can be distinguished)
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and/or
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4. ligate digest LuxCD from plasmid (S+P)
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5. ligate with LuxEG
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PCR for: translado promoter, Pcaa3, LuxAB, VB (psB1C3, psB1K3, psB1A2, pSB1T3)
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So, PCR and electrophoresis for LuxCD and EG (right size).
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New batch of competent cells.
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Ligation LuxCD + LuxEG + psB4K5
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                LuxCD + LuxEG + psB1K3
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New Gibson attempt. sfGFP in psB4K5 (just a try out for new competent cells)
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Transformation in PUC 19
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PUC had colonies. Ligations with colonies. Positive control for Gibson (sfGFP in psB4K5) turn out to be positive :)
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So the problem was: E. cloni instead of TOP 10
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Colony PCR for LuxCDEG in psB4K5. Had red colonies in it.
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PCR for RS1_Kanr_B0015_RS2
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As primers form dimers the PCR was done at more Tm
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amplification of RS1+ Kan + B001 + RS2 --> right size
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Revision as of 11:35, 24 September 2012

Project: Luxilla - Pontificia Universidad Católica de Chile, iGEM 2012