Difference between revisions of "Team:Aix-Marseille/Notebook2"

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

Presentation of our project to the instructors and advisors: they liked it and encouraged us to continue on this way.

Applying for funding: we sent a grant file to the French embassy in USA.

Registration in the iGEM website.

https://www.facebook.com/photo.php?fbid=10206323650749123&set=gm.803648703058628&type=1 A present from the scientific culture unit of the AMU: a new backpack for each team member: we are ready to come to Boston !

Creation of the logo of the AMU team : https://www.facebook.com/photo.php?fbid=10206406746426463&set=gm.809706195786212&type=1

Welcome to Yoann Chabert, a new member of the team!

Presentation of our project to the Institute of Microbiology of the Mediterranean (IMM) for the summer (CNRS - Aix-Marseille Joseph Aiguier)

Welcome to Romain Clément, a new instructor. He is a PhD candidate and his thesis is on CO2 concentration mechanism study in diatoms (Pseudonana)

A new supervisor named Valerie Prima joined our team. The team presented our projet “Chew fight” to the laboratory, they liked it and will support us during the summer.

Our project was published in the local newspaper “Grand Luminy”

Welcome to Ella De Gaullejaque who comes from Paris and joined the team! The game Marseille VS Paris will begin !

Meeting with the whole team (members, advisors, instructors) to finalize the timeline of the experiments. We created a money pot on Leetchi.com to collect money online

The linker between the laccase and the cytochrome has been designed by Wilson. https://www.facebook.com/photo.php?fbid=10207511567166932&set=gm.830198770403621&type=1 The sequences needed were ordered (IDT).

Writing of a newsletter about our project and the synthetic biology. Successful application for being sponsored by the french embassy in USA.

First day at the laboratory, meeting of everyone. Cleaning and room arrangement. https://www.facebook.com/photo.php?fbid=102sa06853955166402&set=gm.834872003269631&type=1

Familiarization and training sessions with technics, supervised by the instructors. We learned basics, as running a PCR, and where we can find everything needed to work in the lab.

Training sessions for PCR

Training sessions for bacteria transformation

Registration for measurement

Training session for Gel purification, PCR clean up, plasmid purification (miniprep)

First purchases of the lab material.

Resuspension of biobricks K863011, K863006, K525998, K844000, E0240, K823005, K823012, I20260 and transformation into E.coli (DH5-alpha).

Negative results (no colony on the plates).

New try for Resuspension of biobricks K863011, K863006, K525998, K844000, E0240, K823005, K823012, I20260 and transformation into E.coli (DH5-alpha).

Positive results (several colonies on the plate).

First meeting with the director of innovations and communications from the cleaning company named ONET.

We talked about our idea and our Chew fight project in order to be sponsored.

Plasmid purification (miniprep) on K863011, K863006, K525998, K844000, E0240, K823005, K823012, I20260 - digestion J23101 et J23115(E/S) et E0240 (X/P) + plasmide (?) I20260 (E/P Dpn1)

Reception of pipettes borrowed from Polytech school. All the pipettes were calibrated

Reception of the microcentrifuge, gel electrophoresis, western blot and SDS-page materials, borrowed from Polytech school.

ONET company presented our project to the Executive office.

Positive answer from ONET: it sponsored our project with 7000 euros !

Resuspension of the biobrick “12” (cyt C Shewanella) received from IDT, ligation into pSB1C3, transformation into DH5-alpha.

Colony PCR on BL21 strain to amplify the 3 parts of ccm operon. Oligos were too concentrated and inhibited the reaction.

Colony PCR and electrophoresis to check the size of the biobrick “12” (cytochrome c Shewanella). Positive result.

Resuspension of biobricks “05”, “08”, “30”, “11” received from IDT. E/P digestion, ligation into pSB1C3, transformation into E.coli (TG1). Negative result (no colony on the plate).

Second try to resuspend the biobricks “05”, “08”, “30”, “11” received from IDT and resuspension of the biobrick “13” received from IDT. E/P digestion, ligation into pSB1C3, transformation into E.Coli (TG1). Positive result (several colonies on the plate).

Second try for Colony PCR on DH5-alpha strain to amplify the 3 parts of ccm operon with diluted oligos at 1/10. Positive result except for part 1: there are some contaminants due to nonspecific hybridization.

Reception of oligos from SIGMA to amplify the laccase of E.coli and T.thermophilus.

Third try for colony PCR on DH5-alpha to amplify part 1 of ccm operon. The Tm changed from 55 to 60°C. Positive result.

Colony PCR and electrophoresis to check the size of the biobricks “05”, “08”, “30”, “11” and “13”. Positive results except for “08” et “11”.

Starters of “05”, “13”, “30”.

Plasmid purification (miniprep) of the biobrick “12”, E/P digestion to check the size of the insert. Positive result.

High fidelity DNA amplification of biobricks “09” and “10” by PCR with the Q5 polymerase in order to remove the codon-STOP. PCR clean up. Negative result (nonspecific amplification).

Reception of the biobrick “15” from IDT.

Colony PCR and electrophoresis to check the size of the biobricks “08” and “11”. Positive result for “08” but not for “11”. Starter of “08”.

New DH5-alpha competent cells done.

Second try for high fidelity DNA amplification of biobricks “09” and “10” by PCR with the Q5 polymerase in order to remove the codon-STOP (the new synthesized biobricks are called respectively “35” and “36”). PCR clean up. Negative result (non-specific amplification). An electrophoresis was done on the total PCR product and was extracted and purified by PCR clean up.

Miniprep of “08”. E/P digestion to check the size of the insert. Negative result (unexpected size).

Resuspension of biobrick “15” received from IDT. E/P digestion, ligation into pSB1C3, transformation into E.coli (DH5-alpha). Negative result (no colony on the plate).

New TG1 competent cells done.

Transformation efficiency test with the transformation efficiency kit of iGEM on the new DH5-alpha competent cells. Positive result (the DH5-alpha cells are competent enough to be transformed with our constructions).

Colony PCR and electrophoresis to check the size of the biobricks “05” and “13”. Positive result. Starters of « 05 » and « 13 ».

SLIC reaction to ligate the 3 parts of ccm operon into the pSB1C3 plasmid. Transformation into E.coli (TG1). Negative result (no colony on the plate).

SLIC reaction to ligate the new biobricks “35” and “36” into pSB1C3. Transformation into E.coli (TG1). Negative result (no colony on the plate).

Transformation efficiency test with the transformation efficiency kit of iGEM on the new TG1 competent cells. Positive result (the TG1 cells are competent enough to be transformed with our constructions)

Miniprep of “05” and “13” and E/P digestion to check the size of the insert.

New try to transform “08” into pSB1C3 into DH5-alpha.

Resuspension of biobricks “09” and “10” from the registry, transformation into TG1.

Construction of “30-02”, “12-02” : E/X digestion of “30” and “12”, S/P digestion of “02”, ligation into pSB1A3, transformation into TG1. Negative result (no colony on the plate).

New try for the SLIC reaction (ccm operon), “35” and “36”. Negative result (no colony on the plate).

New try with different volumes for resuspension of biobricks “15” and “08” received from IDT. E/P digestion, ligation into pSB1C3, transformation into E.coli (TG1). Positive result (several colonies on the plate).

Colony PCR and electrophoresis to check the size of the biobricks”08”, “09”, “10”,”12-02”, “30-02”, “32” and “34”. Positive result for “09” and “10” (expected size). Starters of « 09 » and « 10 ».

Negative result for “32”, “34”, “12-02”, “30-02” (No colony on the plate) and for “08” (unexpected size).<

New try to construct “30-02”, “12-02”: E/X digestion of “30” and “12”, S/P digestion of “02”, ligation into pSB1A3, transformation into TG1. Positive result (several colonies on the plate).

Colony PCR and electrophoresis of “08”, “15”, “12-02” and “30-02” to check the size of the insert. Positive results for “15”, “12-02” and “13-02” (expected size) but negative result for “08” (several sizes observed). Starters of “08”, “15”, “12-02” and “30-02”.

Plasmid purification (miniprep) of “05”, “09” and “10”. Problem encountered: genomic DNA contamination.

E/P digestion and electrophoresis to check the size of the insert of “05”, “09”, and “10”.

Miniprep of “08”, “15”, 12-02”, “30-02”. E/P digestion and electrophoresis to check the size of the insert of “08”, “15”, 12-02”, “30-02”. Positive results for “15”, “12-02”, “30-02” (expected size) and negative result for “08” (unexpected size).

Comment: constructs “12-02” and “30-02” have to be compared with the single “12” and “30” to check if “02” was added.

Colony PCR and electrophoresis to check the size of the insert of “08”. Negative result

Electrophoresis of “12”, “12-02”, “30” and “30-02”. No result (no DNA observed on the agarose gel)

Construction of “09-02”, “10-02” and “13-02”: E/S digestion of “09”, “10” and “13”. X/P digestion of “02”. Ligation into pSB1A3, transformation into TG1. Positive results for “09-02” and “13-02” (1 colony on the plate). Negative result for “10-02” (no colony on the plate).

Resuspension of biobrick “11” and new try for the “05” and the “08” received from IDT. E/P digestion and ligation into pSB1C3. Transformation into TG1. Positive result for “11” (several colonies on the plate) but not for “05” and “08” (no colony on the plate)

Colony PCR and electrophoresis to check the size of the insert of « 11 », “09-02” and “13-02”. Uncertain results (problems: DNA is not observed on the agarose gel). “13-02” seems good: starter of “13-02”.

New try with new oligos for the colony PCR and electrophoresis of « 08 ». No result (no DNA observed on the agarose gel).

New try with new volumes for resuspension of the biobrick “05” received from IDT. E/P digestion and ligation into pSB1C3. Transformation into TG1. Negative result (no colony on the plate).

New try to construct “09-02”, “10-02” and “13-02”. E/S digestion of “09”, “10” and “13”. X/P digestion of “02”. Ligation overnight into pSB1A3, transformation into TG1 (07/31). Positive result for “13-02” (several colonies on the plate. Negative result for “09-02” and “10-02” (no colony on the plate).

E/P digestion to check the size of the insert of “30”, “30-02”, “12”, “12-02”. Results seems good for “12” and “12-02”: send for sequencing. Negative results for “30”, “30-02” (unexpected sizes).

Miniprep of « 13-02 ». E/P digestion and electrophoresis to check the size of the insert. Positive result (expected size).

Colony PCR and electrophoresis to check the size of the insert of “13-02”. Negative result (unexpected size).

Transformation of “09-02”, “10-02” and “13-02” into TG1.

pSB1C3 backbone was running out. New backbone done: E/P digestion on measurement biobricks, electrophoresis and purification of the digested plasmid by gel extraction and PCR clean up.

Problem encountered: no DNA observed on the agarose gel.

Colony PCR and electrophoresis to check the size of the insert of “09-02”, “10-02” and “13-02”. Positive results (expected sizes). Starters of “09-02”, “10-02” and “13-02.

New try for making pSB1C3 backbone: E/P digestion of measurement biobricks, electrophoresis and purification of the digested plasmid by gel extraction and PCR clean up.

Resuspension of the biobrick “07” (cotA) received from IDT. E/P digestion and ligation into pSB1A3, transformation into TG1

Colony PCR and electrophoresis to check the size of the insert on “07” => we got a positive clone, starter of this clone launched

Plasmid purification of “13-02” “09-02” “10-02”

E/P digestion of “05” and “11” and ligation “05” + pSB1K3 and “11” + pSB1K3

Transformation of ligation products into E.coli (TG1)

PCR on “07” and “08” => seems to be unclean with the Q5 high fidelity DNA amplification

X/P digestion of “15”

Overnight ligation “12”+”02”+pSB1A3 and “01”+”15”+pSB1T3

“30-02” and “08” got bad results in sequencing

Transformation of ”01-15” and “12-02” into E.coli (TG1)

Plasmid purification of “09-02” “07” “13-02” “09-02” “10-02” => low concentration of “09-02” but we got good result for “13-02”

Transformation of “12-02” and “01-15” into TG1

PCR clean up “07” “08” “24” ==> low plasmid concentration of “07” and “08” to do again.

Colony PCR of “05” “11” “12-02” “01-15” => good but only for “12-02”

PCR clean up of “08” and “07” → PCR products contaminated → PCR clean up

Transformation of “10-02” and “09-02”→ Wrong size starter relaunched

Ligation “09-02” and “10-02” into pSB1A3 and “01-15” into pSB1T3

Colony PCR of “05” and “01-15” --> no results on this PCR

Transformation of “09-02” “10-02” “01-15” into E.coli (TG1)

Ligation “05” + pSB1K3 and “11” + pSB1K3

Plasmid purification of “12-02” --> Bad results with electrophoresis

For “05” “07” “08” “11” → PCR with Q5 high fidelity polymerase and “PCR clean up”

Preparation of BL21 competent cells

Colony PCR of “01-15” → No results => Ligation with the digestion product “01-15” relaunched

For “05” “07” “08” “11” : Digestion E/S + Ligation pSB1C3 + Transformation into TG1

For “30” : Digestion E/P + Ligation into pSB1K3

For “01-15” : Ligation into pSB1T3

PCR clean up of “28”

Colony PCR of “09-02” “10-02” “05” “07” “08” “11”

Preparation of TG1 competent cells

We used the wrong “02” Biobrick we have to do everything again with the good one → Colony PCR of the “new” “02” → size = ok

Transformation of “30” “01-01” “02” into TG1

Preparation of the backbone pSB1A3

Ligation of “05” “08” “07” “11” into pSB1A3

Colony PCR of “30” “21-17” “23-17” “01-15”

For “35” et “36” : Digestion E/S + ligation into pSB1A3 + transformation into TG1

For “37” “38” : Digestion E/P + Ligation into pSB1K3 + transformation into “supercompetent” cells

Plasmid purification of “02” --> Low concentration of plasmid, starter relaunched

For “02” : Digestion X/P

For “13” : Digestion E/S

Ligation of “35-02” “36-02” “12-02” “13-02” into pSB1A3

PCR on “09” et “10” to remove the stop codon

Transformation of “05” “07” “08” “11” into “supercompetent” cells

Colony PCR on “05” “07” “08” “21-17” “11” “35” “36” “37” “38”

Transformation of “12-02” “13-02” “35-02” “36-02” “36-02” into TG1

PCR clean up of “35” “36”

Digestion E/S of “12” “35” “36” “16” “13”

For “12-02” “13-02” “35-02” “36-02” : Ligation into pSB1A3 + transformation in TG1

For “01-15” : Ligation into pSB1T3 + transformation into TG1

For “16-17” : Ligation into pSB1C3 + transformation into TG1

Colony PCR on “38” “05” “07” “36” ”08” “11”

For “39” “30” : Digestion E/P + Ligation into pSB1K3

Colony PCR on “12-02” “13-02” “39” “36-02” “23-17” “35-02”

Plasmid purification of “36” “38” “07” “05” “35” “11” “37” --> Verification ok for “36” “38” “35” “37”

Colony PCR on “05” “07” “08” “11” “16-17” “21-17” “05” “07” “08” “11” “16-17” “21-17” --> Bad results for all

Plasmid purification of “38” “05” “11” “08” “39” “12-02” “13-02” “35-02” “23-17” “35” “01-15” “16-17” “21-17” --> Problem with the ladder, we cannot read the gel

For “38” “39” “08” “05” : Digestion E/S

For “12” “13” “39” “35” “35-02” “13-02” “12-02” : Digestion X/P

For “38-12” “38-13” “39-02” “38-39” “08-02” “36” “05-02”: Ligation into pSB1A3

For “01-3502” “01-1302” “01-1202” “01-35” : Ligation into pSB1T3

For “21-17” “22-17” : Ligation into pSB1C3

==> Transformation for all ligation products into TG1

Colony PCR of “07” “05” “08” “11” with “pool” technic

Colony PCR of “11” “7” => starters launched on positive clones

Plasmid verification of “05” “07” “08” “11” and “16-17”

“07” and “08” have to be relaunched

Colony PCR on transformants from August 17 => Starters on positive clones launched and the constructions “01-3502” and “38-39” have to be rebuilt

Preparation of the backbone pSB1A3

Ligation of “01-1302” “01-1202” “01-35” into pSB1T3

Starters launched of “05” and “11”

Preparation of the backbone pSB1A3

Sequencing results “12-02” “13-02” “35-02” “01-15” are ok

“08” is bad and “05” couldn’t be sequenced

Plasmid purification of “30” “38-13” “39-02” “08-02” “05-02” “38-12” “11” “07” “16-17”

“38-13” “39-02” “16-17” “38-12” send for sequencing

“30” “08-02” “05-02” “11” “07” are bad we cannot use its

Colony PCR of “05-02” “01-1302”

Plasmid purification of “05” and “11” and of 2 starters to make some backbones (pSB1A3 and pSB1T3)

The digestion to prepare pSB1T3 is bad, starter relaunched

Ligation of “38-39” “11-02” into pSB1A3

“38-11” into pSB1C3

“01-1202” “05-02” “01-36” “01-1302” “01-3502” “01-35” “21-17” “22-17” into pSB1K3

Transformation of previous ligation products into DH5-Alpha for “21-17” and “22-17”, into TG1 for “11-02” “38-11” “05-02” and “38-39” and into BL21 for “01-1202” “01-36” “01-1302” “01-3502” and “01-35”

“38-13” “39-02” “11” and “38-12” sent for sequencing

Sequencing result of “05” => bad

Transformation of “18-17” “19-17” “20-17” and “23-17” into DH5-Alpha

Colony PCR of “07”

Digestion E/S fof “16” “38” “11” “22”, X/P for “17” “39” “02” “11” “35”

Ligation of “16-17” “22-17” + pSB1C3 “38-39” “11-02” “38-11” + pSB1A3

“01-35” “05” “07” “08” “30” + pSB1K3

Transformation of “30” “08” “07” “05” into C2987 (“supercompetent” cells)

Transformation of “38-39” “11-02” “38-11” and “01-35” into TG1

Plasmid purification of “01-1202” “01-3502” “05” “01-1302” “07” and of pSBT3 to get some backbone.

Colony PCR of “08” “01-3502” “01-1202” “01-1302” “01-36”

We got many bad results today so bad luck, but we got some good results also.

Colony PCR of “38-39” “38-11” “11-02” “01-35”

Starters launched of positive clones

Transformation of overnight ligation produtcs (“16-17” and “22-17” from August 21)

Plasmid purification to make some pSB1A3 and pSB1T3

First protein expression test, we maybe succeed to express our first laccase

Colony PCR of “16-17” and “22-17” launched overnight => green colonies!!

Starters launched of positive results

Plasmid purification of “22”-17” “16-17” and ‘01-35” => all results are good!!!!

Digestion E/S for “38” and X/P for “39”, ligation overnight into pSB1A3

Transformation of “30” “08” “07” “11” “05” into TG1

Plasmid purification of “16-17” “01-1302” “01-1202” “22-17” “01-3502”

Colony PCR of “05” “07” “08” “11” => no positive results!

Q5 PCR of “05” “07” “08” “11” “30” => positive results

“05” and “01-35” sent for sequencing

Transformation of “05” “07” “08” “11” “30” into TG1

Plasmid purification of “01-36” “19-17” “18-17” “23-17” “20-17” => good results

“01-1202” “01-1302” “01-3502” sent for sequencing

Sequencing results of “01-35” “38-12” “38-13” “39-02” => good results “05” “11” => bad results

Starters launched of “39” “01-1302” “01-1202” “01-3502” “01-15” “13-02” “12-02” “38”

Protein expression => the cytochrome c and the laccase are expressed.

Isolation and starter launched of “32”

Plasmid purification of “39” “01-1302” “01-1202” “01-3502” “01-15” “13-02” “12-02” “38” “16-17”=> no results!

Ligation of “40” “41” “42” “43” “44” “45” “46” “47” “48” “22-17” + pSB1C3

“30” + pSB1K3

Transformation of “30” “40” “45” “48” into TG1

Transformations didn’t work! => “40” “45” “48” relaunched

Sequencing results “01-1302” => without promoter

“01-1202” and “01-3502” are good

Colony PCR of “40” “45” “48”

Starters launched of “18-17” “19-17” “20-17” “21-17” “22-17” “23-17” “32” “33” “01-24” each in triplicate

Stock of backbone pSB1C3 done

Preparation of SDS-PAGE, of solutions and of the column to make the protein purification

Digestion E/P of “22-17” “43” “44” “47” and digestion X/P of “1302” “3902”

Ligation “43” “44” “47” into pSB1C3

“013002” into pSB1K3

“01-3902” “01-1302” into pSB1C3

Colony PCR of “40” “45” “48” all clones are positive, starters launched

Protein purification of “01-3502” “01-1202”

SDS-PAGE of “01-3502” “01-1202”

01-3502 purified and pure!!!! We got our first protein which is a laccase from E.coli

Starters launched of “38” “13-02” “01-36” “01-15” “01-3502” “01-1202” “37” “35-02” “12-02” “35” “36” “01-35” “39” “40” “45” “48”

Plasmid purification of “01-36” “40” “39” “48” “45” “37” “36” “01-1202” “01-36” “01-15” “35-02” “35” “38” “13-02” “12-02” “01-3502” “01-35”

Protein expression of 01-3502 and 01-1202

Colony PCR of “01-15” beause we have doubt concerning colonies => all colonies were positive

Ligation of “43” “44” “47” “03002” “01-1302” “013902” into pSB1C3

Transformation of “43” “44” “47” “013002” into C2987 and “01-1302” “01-3902” “3813-02” “3812-02” into TG1

Verification digestion on “40” “38” “01-15” “48” “45” “01-36” “01-1202” “01-36” “37”

Plasmid purification of “01-1202”

Colony PCR of “3812-02” “3813-02” “01-3902” “01-1302” starters launched on positive results

No colony for “43” “44” “47” “013002”

Preparation of the backbone pSB1C3

Transformation of “013002” “41” “44” “47” into C2987

Plasmid purification of “45” “01-36” “381302” “38” “01-1202” “3812-02” “3813-02”

Colony PCR of “47” “01-3002” “43” “44” => almost everything is positive!!! Great day!

Plasmid purification (miniprep) of “44” “47” “43” “38” “01-1302” “01-1202” “01-3002” “45”

“01-1302” “01-36” “3813-02” seems good so they are send for sequencing.

Construction : Digestion E/S of “38” “01-36” “01-35” “01” and Digestion X/P of “44” “47” “43” “48” “44” “45” “1302” “3813-02” “39-02” “40” “3812-02”

Starter launched on “01-1202” “01-3002” for production and on “0136” for miniprep

Ligation in pSB1C3 of “0136-02” “01-381302” “01-381202” “01-3902” “01-1302” “38-3902” “0136-40” “0136-45” “0136-48” “0135-43” “0135-44” “0135-47”

Transformation of “01-3002” in BL21, of pSB4K5 and pSB3T5 in TG1 and transformation of ligation product in TG1

Purification of “01-1202” (1) and (2):

Induction by IPTG

Storing the cells at -80°C

Colony PCR of “013002” “013002” => all results are positive

Production of “01-1202” : Sonication of cells, centrifugation 10 min at 4500 rpm, ultracentrifugation 45 min at 4500 rpm, solubilizing membrane in Triton 1% during 2h and incubation overnight.

Preparation of SDS Page 15% and a western blot with products of yesterday => results are not good so we have to resart the production

Culture of “01-1202” and “01-3002” in anaerobic condition, cells were centrifuged 10 min at 4500 rpm and pellet were stored at -20°C

A western blot was done → Production of Cytochrome, monomer in the membrane and dimer in the cytoplasm

The next step is to purify “01-1202” and “01-3002” and start a culture on 01-3502 in anaerobic condition