Team:Aix-Marseille/Notebook2
CALENDAR
Suspension of 4 BioBricks:
-E0240 -K823005 -K823012 -I20260
Transformation of E.coli (DH5-Alpha) with biobricks E0240, K823005, K823012 and I20260
Plasmid purification of the 4 BioBricks transformed on June 26, using a Promega kit.
Plasmid digestion of the BioBricks:
E0240 => X/P
K283005 => E/S
K823012 =>E/S
I20260 => E/P
Suspension of BioBrick J23119 and transformation into E.coli (TG1)
Observation of transformants.
Digestion of pSB1K3 : E/P + Dpn1
Electrophoresis of the digested backbones, E0240 K823005 and K823012
We got good results => ligation:
K823005 + E0240 + pSB1K3 (Named M1)
K823012 + E0240 + pSB1K3 (Named M2)
I20260 + pSB1K3 (named M3)
Transformation of these ligations and a plasmid from our lab =LismA201 (which is a GFP with promotor, RBS and terminator)
Suspension of BioBricks -I13504 -J23100 -J23118 -J23106 -J23117 -J23103
Transformation of these BioBricks into E.coli (TG1)
Plasmid purification of J23119 => bad results (due to the Macherey-Nagel kit?)
Preparation of E.coli (TG1 and DH5-alpha) competent cells
Observation of transformation results
PCR on recombinant clones => LismA201/M1/M2 and M3
TheBioBrick J23100 was not the BioBrick we wanted we made a little error in the suspension (bad plate)
Transformation of the correct J23100 into E.coli (TG1)
Test with the Macherey-Nagel kit => it works!!
Starters of positive clones verified by PCR and of -I13504 -J23100 -J23118 -J23106 -J23117 -J23103
Plasmid purification of starters from positive clones of M1/M2/M3 and LismA201 and of -I13504 -J23100 -J23118 -J23106 -J23117 -J23103
E/S digestion of -J23118 -J23106 -J23117 and J23103
XP digestion of -I13401 and I13504
Ligations:
- J23101 + I13504 + pSB1C3
- J23118 + I13504 + pSB1C3
- J23106 + I13504 + pSB1C3
- J23117 + I13504 + pSB1C3
- J23103 + I13504 + pSB1C3
- K525998 + I13401 + pSB1C3
Dosage of these purified plamids
Digestion E/P on purification products and electrophoresis to detect positive ligations
Suspension of BioBrick J23100, we did an error when we suspended the previous BioBrick
Ligations:
- BBa_K823005 + I13504 + pSB1K3
- BBa_J23118 + I13504 + pSB1K3
- BBa_K823008 + I 13504 + pSB1K3
- BBa_K823013 + I13504 + pSB1K3
- BBa_J23103 + I13504 + pSB1K3
- BBa_K525998 + I13401 + pSB1K3
Transformation of ligation products
PCR on some M1 clones.
Test of competent cells BL21 and DH5-Alpha with the iGEM test kit.
Digestion E/P of positive clones M1/M2/M3/LismA201 and electrophoresis.
Plasmid purification of J23100, DNA dosage, E/P digestion and electrophoresis
PCR of ligation products
- BBa_K823005 + I13504 + pSB1K3
- BBa_J23118 + I13504 + pSB1K3
- BBa_K823008 + I 13504 + pSB1K3
- BBa_K823013 + I13504 + pSB1K3
- BBa_J23103 + I13504 + pSB1K3
- BBa_K525998 + I13401 + pSB1K3
- And PCR M1 relaunch
Starters launched of positive clones
PCR on BBa_K823008 + I 13504 + pSB1K3 and on J23100 + I13504 + pSBK3
Transplanting of:
- J23100 + I13504 + pSB1K3
- K823008 + I13504 + pSB1K3
- I13504 + pSB1C3
Starters of:
- BBa_K823008 + I 13504 + pSB1K3
- J23100 + I13504 + pSB1K3
- M1
Plasmid purification of
- BBa_K823005 + I13504 + pSB1K3
- BBa_J23118 + I13504 + pSB1K3
- BBa_K823013 + I13504 + pSB1K3
- BBa_J23103 + I13504 + pSB1K3
- BBa_K525998 + I13401 + pSB1K3
Digestion E/P of :
- BBa_K823005 + I13504 + pSB1K3
- BBa_J23118 + I13504 + pSB1K3
- BBa_K823013 + I13504 + pSB1K3
- BBa_J23103 + I13504 + pSB1K3
- BBa_K525998 + I13401 + pSB1K3
- M1/M2/M3/LismA201
M1 has to be done again
Plasmid purification of
- J23100 + I13504 + pSB1K3
- K823008 + I13504 + pSB1K3
- and M1
Digestion E/P of purified plasmids
Electrophoresis and sequencing of positive results
PCR on transformants of J23103 + I13504 + pSB1K3
Starters of positive clones
Digestion E/P of
- J23100 + I13504 + pSB1C3
- J23101 + I13504 + pSB1C3
- J23118 + I13504 + pSB1C3
- J23106 + I13504 + pSB1C3
- J23117 + I13504 + pSB1C3
- J23103 + I13504 + pSB1C3
- K525998 + I13401 + pSB1C3
Plasmid purification of two J23103 + I13504 + pSB1C3 starters
Digestion E/P of the two J23103 + I13504 + pSB1C3 purified plasmids
Ligation in pSB1C3
Transformation into E.coli (DH5-Alpha) of:
- J23100 + I13504 + pSB1C3
- J23101 + I13504 + pSB1C3
- J23118 + I13504 + pSB1C3
- J23106 + I13504 + pSB1C3
- J23117 + I13504 + pSB1C3
- J23103 + I13504 + pSB1C3
- K525998 + I13401 + pSB1C3
PCR on many transformants:
- J23100 + I13504 + pSB1C3
- J23101 + I13504 + pSB1C3
- J23118 + I13504 + pSB1C3
- J23106 + I13504 + pSB1C3
- J23117 + I13504 + pSB1C3
- J23103 + I13504 + pSB1C3
- K525998 + I13401 + pSB1C3
Electrophoresis of the PCR products from July 20th
Starters on positive clones
Transformation of J23117 + I13504 + pSB1C3 because it was negative
PCR of J23117 + I13504 + pSB1C3
Starters on positive clones
The sequences are bad we have to do it again except for M3 which was the only good one
Starter of I13504 to make a stock of positive control
Plasmid purification of
- J23100 + I13504 + pSB1C3
- J23101 + I13504 + pSB1C3
- J23118 + I13504 + pSB1C3
- J23106 + I13504 + pSB1C3
- J23103 + I13504 + pSB1C3
- K525998 + I13401 + pSB1C3
Digestion E/P and electrophoresis to see if the insertion is good
We took the wrong BioBrick!!!
Transformation of the right I13504 into E.coli (TG1), two starter cultures launched
DNA purification of I13504. Digestion X/P of I13504
Digestion E/S of J23100, K823005, J23118, K823008, K823013
Ligation:
- BBa_J23100 + I13504 + pSB1C3
- BBa_K823005 + I13504 + pSB1C3
- BBa_J23118 + I13504 + pSB1C3
- BBa_K823013 + I13504 + pSB1C3
- BBa_J23103 + I13504 + pSB1C3
- BBa_K525998 + I13401 + pSB1C3
But we did not get enough pSB1C3 only BBa_J23100 + I13504 + pSB1C3
BBa_K823005 + I13504 + pSB1C3 could be launched
Transformation of BBa_J23100 + I13504 + pSB1C3
BBa_K823005 + I13504 + pSB1C3 into E.coli (TG1)
PCR of BBa_J23100 + I13504 + pSB1C3 and BBa_K823005 + I13504 + pSB1C3
Preparation of pSB1C3 Backbone with a digestion E/P of BBa_K525998 electrophoresis and gel purification of the highest band
Ligation:
- BBa_J23118 + I13504 + pSB1C3
- BBa_K823013 + I13504 + pSB1C3
- BBa_J23103 + I13504 + pSB1C3
- BBa_K525998 + I13401 + pSB1C3
Transformation into E.coli (TG1)
- BBa_J23118 + I13504 + pSB1C3
- BBa_K823013 + I13504 + pSB1C3
- BBa_J23103 + I13504 + pSB1C3
- BBa_K525998 + I13401 + pSB1C3
- M1/M2
PCR on a green colony seen in the “magic box” => no positive results
Thermal shock forgot for the transformations, we have to do it again!!
PCR of J23100 + I13504 + pSB1C3 and BBa_K823005 + I13504 + pSB1C3
Starters launched on positive results
Transformation into E.coli (TG1)
- BBa_J23118 + I13504 + pSB1C3
- BBa_K823013 + I13504 + pSB1C3
- BBa_J23103 + I13504 + pSB1C3
- BBa_K525998 + I13401 + pSB1C3
- M1/M2
Plasmid purification of J23100 + I13504 + pSB1C3
E/P digestion of J23100 + I13504 + pSB1C3 and electrophoresis to see the insertion of the BioBrick
Preparation of starters to use the TECAN, a plate reader we use to mesure GFP
We learned how to use the TECAN
PCR on M1, M2, BBa_K823013 + I13504 + pSB1C3, BBa_K823013 + I13504 + pSB1C3
Transformation of BBa_K823005 + I13504 + pSB1C3 into E.coli (TG1)
Ligation overnight:
- BBa_J23118 + I13504 + pSB1C3
- BBa_K823013 + I13504 + pSB1C3
- BBa_J23103 + I13504 + pSB1C3
- BBa_K525998 + I13401 + pSB1C3
Transformation of ligation products:
- BBa_J23118 + I13504 + pSB1C3
- BBa_K823013 + I13504 + pSB1C3
- BBa_J23103 + I13504 + pSB1C3
- BBa_K525998 + I13401 + pSB1C3
PCR on the only clone of BBa_K823005 + I13504 + pSB1C3 we got => positive result=> starter launched
Plasmid purification of BBa_K823005 + I13504 + pSB1C3 and E/P digestion to see the right insertion of the insert
PCR on green colonies on “the magic box”
PCR of BBa_K823005 + I13504 + pSB1C3 done again because of bad results
Starter launched on positive results
Starter of BBa_K823013 + I13504 + pSB1C3 and on BBa_K823008 + I13504 + pSB1C3 launched
J23100 + I13504 + pSB1C3 good sequencing!!!
Ligation overnight BBa_J23101 + I13504 + pSB1C3
Transformation of J23101 + I13504 + pSB1C3 into E.coli (TG1)
BBa_K823008 + I13504 + pSB1C3 prepared to send for sequencing
PCR on J23101 + I13504 + pSB1C3
Starter of positive clones
Ligation J23103 + I13504 + pSB1C3
Transformation of J23101 + I13504 + pSB1C3 into E.coli (TG1)
Plasmid purification of J23101 + I13504 + pSB1C3 starters
J23101 + I13504 + pSB1C3 prepared to send for sequencing
Starter of BBa_K823013 + I13504 + pSB1C3 launched => preparation for sequencing
Isolation of BBa_K823008 + I13504 + pSB1C3
X/P digestion of I13504
Ligation BBa_K823008 + I13504 + pSB1C3
BBa_K823005 + I13504 + pSB1C3 has the right sequence!
Ligation J23103 + I13504 + pSB1C3
Transformation of J23103 + I13504 + pSB1C3 into E.coli (TG1)
Send for sequencing BBa_K823008 + I13504 + pSB1C3 and BBa_K823013 + I13504 + pSB1C3
Plasmid purification of BBa_K823013 + I13504 + pSB1C3
BBa_K823013 + I13504 + pSB1C3 send for sequencing
Starter launched on positive clones from the PCR done on August 16
Plasmid purification and digestion E/P to check the insertion => wrong results
Try again on the same clone
Ligations: BBa_K823008 + I13504 + pSB1C3 and BBa_K823013 + I13504 + pSB1C3
BBa_K823008 + I13504 + pSB1C3 and BBa_K823013 + I13504 + pSB1C3 and J23100 + I13504 + pSB1C3 send for sequencing
J23103 + I13504 + pSB1C3 has a good sequencing result
BBa_K823008 + I13504 + pSB1C3 and BBa_K823013 + I13504 + pSB1C3 => good sequencing result
Transformation of ligation products of J23119 + I13504 + pSB1C3 and BBa_K823013 + I13504 + pSB1C3
Transformation of J23119 + I13504 + pSB1C3 and K823013 + I13504 + pSB1C3 into E.coli (DH5-Alpha)
First try of construction of a box to observe GFP called “magic-box”. First try with coomasie blue as light filter, public lab spectrometer, yellow filter and a webcam
J23119 + I13504 + pSB1C3 (obtained the August 24th) send for sequencing
Construction of the magic-box without the public lab spectrometer
Coomasie blue concentration determination and first assay with the magic box
Sequencing results of J23119 + I13504 + pSB1C3 => bad
K823013 + I13504 + pSB1C3 clone 5 => good
K823013 + I13504 + pSB1C3 clone 2 => bad
Isolation of K525998 + I13401 + pSB1C3, K823008 + I13504 + pSB1C3, J23119 + I13504 + pSB1K3 and K823013 + I13504 + pSB1K3
E/P digestion of J23119 + I13504 + pSB1K3
Ligation overnight:
J23119 + I13504 + pSB1C3
tarter launched on another J23119 + I13504 + pSB1C3 clone
Measure of fluorescent intensity with different bacterial OD
Determination of the medium that will be use => LB or PBS1X
Ligation K823013 + I13504 + pSB1C3
Transformation of K823013 + I13504 +pSB1K3 into E.coli (DH5-Alpha)
Test on many machines to observe GFP => confocal microscope, magic-box, TECAN, and fluorimeter
Transformation of K823013 + I13504 +pSB1C3 gives no transformants
Measure of GFP intensity with TECAN and confocal microscope on “18-17”, “19-17”, “20-17”, “21-17”, “22-17”, “23-17”, “01-24”, “33” and “32”
Measure of GFP intensity with fluorimeter and magic box on “18-17”, “19-17”, “20-17”, “21-17”, “22-17”, “23-17”, “01-24”, “33” and “32”
Sequencing result: “23-17” is bad
