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