Team:Paris Bettencourt/Notebook/Riboswitch

July 27th

Goal

E coli DH5-alpha transformation with RFP from iGEM plate 4 - 2H (pSB1A3 backbone)

Procedure

  1. I took iGEM plate 4 - 2H (RFP, BBa_J04450 in PSB1A3 backbone) and resuspend it in 10 uL of distilled water.
  2. Used E coli competent cells from NEB 2 tubes c2987 (50uL) : 1 control (C) and one for transformation (T)
  3. Introduced 2 uL (200-300pg/ul) of my suspended plasmid solution in T.
  4. Waiting 30 min in ice
  5. Heat shock 30 sec 42°C
  6. Then 5min in ice
  7. Add 200uL SOC medium
  8. Recovery 2 hours then plate it on 3 different 100ug/mL Ampicillin LB
  9. Incubate 37°C till tomorrow

Results

The day after I had colonies growing, nothing on my control
    I took 2 different colonies and do a 8 hours culture in two different tubes T1 an T2 in LB + Amp 100ug/mL, in order to do a glycerol stock.


July 29th

Goal

Making chemically competent cells

Procedure

I followed the following method
  • The day before, start an overnight Liquid Culture of the E. coli strain. (5mL of LB + Antibiotics + Single colony)
  • The next day, set the Centrifuge at 4°C and place on ice a tube containing 15mL of 100mM CaCl2 and a tube containing 4mL of 100mM CaCl2 + 15% Glycerol.
  • In an Erlenmeyer, add 50mL of LB and 250uL of the Liquid Culture, (for quicker growth, use a larger Erlenmeyer)
  • Put the Erlenmeyer to shake at 37°C until the OD(600) reaches 0.4 - 0.5.
  • When the OD(600) is reached, transfer in a 50mL Falcon tube and centrifuge 10min at 4°C.
  • Remove the supernatant and resuspend in 15mL of ice cold 100mM CaCl2. -Leave on ice for 4h.
  • Centrifuge 10min at 4°C.
  • Remove the supernatant and resuspend in 4mL of ice cold 100mM CaCl2 + 15% Glycerol.
  • Aliquot in 1.5mL eppendorf tubes and store at -80°C until use.


    • July 30th

    Goal 1

    PCR my G block and backbone

    Procedure

  • PCR1 = Backbone + 068 and 069 primers, before purify
  • PCR2 = FINAL + 068 and 069 primers, before purify
  • Then PCR purification
  • Nanodrop : PCR 1 = 61ng/uL ; PCR2 = 25,4 ng/uL

    • Goal 2

    Digestion/Ligation/Transformation

    Procedure

    First, digestion :
  • PCR1 ADN : 40uL = 2,4 ug 4uL EcoRI, PstI, FastAp 12 uL FD buffer 56 uL water TOT =120uL
  • PCR2 ADN : 40uL = 1ug 6 uL EcoRI, PstI 12 uL FD buffer 64 uL water TOT= 128uL
  • 10 min at 37°C

    • Then PCR purification
  • Nanodrop : nanodrop : PCR 1 = 20,7 ng/uL ;PCR2 = 25,7

    • For the ligation :
  • Vector PCR1 100 ng = 5 uL; Insert PCR 2 300 ng = 12uL T4 ligase buffer 2uL ; T4 ligase 1 uL ; Total 20 uL
  • 20-40 min on the bench

  • Nanodrop : L= 270 ng/uL

    • Then I did a heat shock transformation (following the iGEM protocol : http://parts.igem.org/Help:Protocols/Transformation )
  • C + = 10 ng/uL of RFP in pS1C3, 2 uL transformed (10pg/ul RFP Control (pSB1C3 w/ BBa_J04450)
  • C - = nothing but efficients cells
  • T = Plasmid construct 2uL transformed
  • Each with 50 uL of chemically competents cells done yesterday.

    • Then I did 5 different plates :
  • 5 plates with 25ug/mL chloramphenicol in LB
  • 200 uL control + , 20 uL control +
  • 200 uL T , 20 uL T
  • 200 uL C -
  • Incubation overnight at 37°C


    • July 31th

    Goal 1

    Check the transformations I've done yesterday

    Procedure

    According to my plates my competent cells are working, red colonies on the C+. Transformation of my construction didn’t worked, I’ll start again all the process : PCR, purify, digest, ligate, integrate (heat shock), overnight culture.

    Goal 2

    PCR my backbone and my insert

    Procedure

  • PCR1' = Backbone + 068 and 069 primers, before purify
  • PCR2' = FINAL + 068 and 069 primers, before purify
  • After PCR and after purification : 1'p = 66,5 2'p =90,5 ng/uL
  • The gel migration shows a bad result for PCR 1’ (Linearized pSB1C3)


    • August 1st

    Goal

    PCR my backbone and my insert again : 1" and 2"

    Procedure

    PCR then purify

    August 5th

    Goal

    Ligation/transformations (insertion of FINAL block from IDT in the pSB1C3 backbone, FINAL is my Riboswitch associated with an EGFP)

    Procedure

  • Used 3 different insert PCR products
  • Ligation after digestion and purification of PCR2’ = 94,6ng/uL , PCR2’’= 57,5 ng/uL , PCR2’’’=83,4 ng/uL ; Backbone pSB1C3 = 37,9ng/uL
    • ' " "'
    Insert 4 7 5
    Backbone 5 5 5
    Buffer 2 2 2
    Ligase 1 1 1
    Water 8 5 7
    TOTAL 20uL 20uL 20uL
    I did then a transformation using the iGEM protocol (Heat Shock) with 50uL of chemically competent cells + 3 uL of Ligation Product for each (pSB1C3-TOTAL)
  • C + : pSB1C3-RFP
  • C - : just competent cells

  • Plated on Cm 25 ug/mL
  • 5 plates : ‘,’’,’’’,C+,C-

    • Results

  • C- : nothing
  • C+ : lot of red colonies
  • ‘ : lot
  • ‘’ : lot
  • ‘’’ : lot
  • I should have done a control transformation with just the backbone


    • August 6th

    Goal 1

    Cultivate my transformants

    Procedure

  • There is red and white transformant colonies, all resistant to Cm 25
  • I selected some white colonies overnight culture
  • On plate LB Cm —> it grew (some red, maybe contamination)
  • On broth LB Cm —> it grew (some red, maybe contamination)
  • Inoculated at 4:00 pm in LB Cm 25 bacteria from a culture ( red —> RFP ) with different AdoCbl concentrations in order to detect the minimal amount of B12 viable for e.coli : 5; 10; 20; 40; 80; 160 ng/mL

    • Goal 2

    PCR the pSB1C3 backbone and my FINAL riboswitch construction

    Procedure

  • 2 PCR each
  • I1 and I2 using primers 150 and 151
  • LP1 and LP2 using primers 152 and 153
  • Results : Gel migration good only for I2 at 1300 bp.
  • Purification then nano drop : good for I2

    • Results

    Gel migration :