<p>New try for Resuspension of biobricks K863011, K863006, K525998, K844000, E0240, K823005, K823012, I20260 and transformation into E.coli (DH5-alpha). </p>
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<p>Transformation of J23101 + I13504 + pSB1C3 into <i>E.coli</i> (TG1)</p>
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<p>Positive results (several colonies on the plate).</p>
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<p>BBa_K823008 + I13504 + pSB1C3 prepared to send for sequencing </p>
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
Transformation BBa_K823008 + I13504 + pSB1C3 into E.coli (TG1)
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
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
“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
“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
