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Team:Lyon-INSA-ENS/Realisation/Week10
From 2011.igem.org
Week 10
From Monday the 22th of August to Friday the 26th of August 2011
Monday
Transformations and controls for future tests
QIAGen extraction of NM522 and NM522+pIG4 and electrophoresis : no plasmid is in the strains.
A new NM522 strain is found in another lab.
Plating of 10µL of this new NM522 on LB
Transformation and plating of NM522 on LB with : pUC18 (=pIG6) (3 µL) + Amp, pIG25 (3 µL) + Cm
Transformation and plating of S19 on LB and the antibiotic Amp with : p157 (3 µL) + SpcR, p115 (3 µL) + SpcR, p127 (3 µL) + Kn, p116 (3 µL) + Kn
Results of Adherence Test - Result of Flocculation Test
Revealing of 24 well plates and flocculation test from 08.19.11. The (2) has failed. The others plates are quite good.
Re-synthetising csg-BAEFG with standard iGEM restriction sites
Digested the PCR result with both EcoRI and PstI.
Cloned the resulting DNA in pSB1C3.
Transformed the result in NM522 strain.
Creating the rcn-ompR234 part
Digested both our rcn part and the ompR part from last year's INSA iGEM team with respectively EcoRI, SpeI and XbaI, PstI.
Digested pB1C3 with both EcoRI and PstI.
3A ligation on those construct.
Transformation of a NM522 strain with the result of the ligation.
Others
Both NM522 and NM522/pIG24 grew on LB+Amp ( liquid and solid ) : antibiotic concentration in liquid was not sufficient, a more concentrated solution of Amp (10mg/mL which corresponds to 100X) is made and sterilized by filtration.
Creation of our own LB+Amp plates by plating 200µL of Amp on a LB plate.
Plating of several AmpS strains on the previous plates : PHL818, MC4100, NM522+pIG4, NM522 from the collection, NM522 from the previous LB+Amp plate, a new NM522 found in another lab.
Dissolution of 0.08g of Spectinomycin ( C14H24N2O7 + 2 HCl + 5 H20 ) into 5mL water and filtration to obtain a solution at 10mg/mL.
Tuesday
Transformations and controls for future tests
Start of 5mL liquid cultures of two clones for each plates obtained by the previous transformations, in order to check the presence of the right plasmids and start fluorescence tests.
Transformation and plating of NM522 on LB with : p157 (2 µL) + Spc, p115 (2 µL) + Spc, p56 (2 µL) + Amp, p10 (3 µL) + Amp, pIG30 (2 µL) + Cm, p127 (2 µL) + Kn, p116 (5 µL) + Kn
Strain Collection
Storage of S20.
Plating of S20 on home-made LB+Amp dishes and start of 5mL liquid cultures in LB+Amp.
Start of 50mL liquid culture of NM522/piG6 for a future extraction.
Storage of NM522/piG6 in the collection as S21.
Adherence Test Preparation
Start of 5mL cultures for 24 well plates in M63G medium, with 50µL antibiotic if appropriate, from 10µL of saturated cultures : PHL818, NM522, NM522+pUC18 (Amp), S18(Amp).
Re-synthetising csg-BAEFG with standard iGEM restriction sites
Miniprep on the NM522 growing overnight, results are unclear.
Creating the rcn-ompR234 part
Miniprep on 3 different clones, analysed on gel, one seems to be correct.
Supposedly correct clone, and DNA extracted from it stored in collection.
Others
Results of the LB+Amp tests from the previous day : MC4100, NM522+piG4, PHL818, the new NM522 don't grow.
NM522 and NM522 from the collection produce a few colonies -> the collection was contaminated by an AmpR strain.
NM522 in the collection (S11) is replaced by the new NM522.
Test of 2 other batches of LB+Amp dishes by plating the new NM522 on them.
Plating of NMYO on LB+Kan from an old solid culture.
Wednesday
Transformations and controls for future tests
Start of 5mL liquid cultures of two clones for each plates obtained by the previous transformations, in order to check the presence of the right plasmids.
Plating of MC4100 on LB from an old solid culture (07/26) for future transformations.
Miniprep of :
- S19/p157
- S19/p115
- S19/p127
- S19/p116
Digestion by E and electrophoresis to control the presence of the two plasmids : the plasmid piG16 doesn’t seem to be in these strains!!
Plasmid Collection
Midiprep of pIG6 (=pUC18)
Digestion by E and electrophoresis to control the plasmid : one band at 2.5-3kb which is coherent.
Nanodrop quantification : c=83ng/µL, 260/280=2.02
Storage of 300µL of the plasmid at -20°C.
Start of 50mL cultures (500µL antibiotic if necessary and 100µL bacteria ) of NM522 + p10 (Amp),NM522 + p127 (Kan),NM522 + p56 (Amp),NM522 + p157 (Spc),NM522 + p115 (Spc)for future extractions.
Adherence Test - pRcn-csgBAEFG Characterization
Start of 24 well plate adherence test in M63G medium with PHL818 (positive control), NM522 (negative control), S21+Amp without Co, S21+Amp+Co 0,1mM, S18+Amp without Co, S18+Amp+Co 0,1mM.
Incubation at 30°C for 48h.
Fluorescence Tests
Measuring the fluorescence and the OD600 of the tubes from the transformation of tuesday.
Re-synthetising csg-BAEFG with standard iGEM restriction sites
New minipreps on new NM522 clones results are still unclear. Transformation might not have worked.
Others
Result of the test of the 2 batches of LB+Amp : NM522 doesn't grow so they seem efficient.
Thursday
Transformations and controls for future tests
Start of 5mL liquid culture of MC4100 from solid culture (08/24).
Start of 5mL liquid culture for fluorescence of : S19/p127, S19/p116, NM522/p127, NM522/p116 in LB/2 and M63G : 30°C and slow agitation
Start of 5mL liquid culture for fluorescence of : S19/p115, S19/p157, NM522/p115, NM522/p157 in LB/2 and M63G : 37°C and fast agitation. For each strain, there are 4 tubes without Cobalt and with 3 concentrations of Cobalt
Transformations and plating of MC4100 on LB+Amp with : piG6 (3 µL), piG2 (3µL) and sterile water (3µL) for negative control.
Plasmid Collection
Midiprep of p56 and p157.
Digestion by E and electrophoresis to control the plasmid : the obtained bands are coherent for each plasmid.
Nanodrop quantification :
-p56: c = 15,1 ng/µL
-p157: c = 19,3 ng/µL
Storage of 300µL of the plasmid at -20°C.
p56 = piG31
p157 = piG32
Start of 50mL liquid cultures (500µL antibiotic if necessary and 100µL saturated cultures (08/24)) of NM522/piG30 (Cm), NM522/p127 (Kan), NM522/p116 (Kan) for future extractions.
Others
Pouring of Cm+Amp dishes
Preparation of new Spc at 10mg/mL from 0.15g ( C14H24N2O7 + 2 HCl + 5 H20 ) into 10mL water.
Friday
Transformations and controls for future tests
Digestion of R1, R2, C1, N1, N2 minipreps by E and electrophoresis :
R2 : 2kb, not good
R1 : 2.8kb, validated.
N2 : 2kb, 5kb , not good
N1 : 8kb , not good
C1 : 3kb, 2.5kb , not good
Plating on LB+Amp of two clones of each transformation (08/25): MC4100/piG6 and MC4100/piG2.
Miniprep of two clones of : S19/p115, S19/p116, S19/p127, S19/p157, NM522/p115, NM522/p116, NM522/p127, NM522/p157, NM522/p10 to control then the strains we are using for fluorescence tests.
Plasmid Collection
Midiprep of: p10, p115, p127, p116 and piG30 (transformed in NM522) for storage in plasmid collection after control.
Flocculation Test
Start of a flocculation test in two media LB/2 and M63G:
-5mL of medium
-50µL of Amp
-One clone of the transformation plates (08/25): MC4100/piG2 and MC4100/piG6 (negative control)
-Co 10µM for MC4100/piG2
These liquid cultures are let over the weekend at 30°C and low stirring (70).
Results of Fluorescence Tests
Measuring the fluorescence and the OD600 of all the liquid cultures started on Thursday.
Results of Adherence Test - pRcn-csgBAEFG Characterization
Revealing of the 24 well plates started on Wednesday.
Measuring OD600 and Crystal Violet coloration.
Adherence Test
Start of 24 well plates :
(1) in M63G : NM522 (negative control),PHL818 (positive control),NM522/piG3 (without cobalt and with 0.1mM cobalt),NM522/p10 (without cobalt and with 0.1mM cobalt),S19(without cobalt,with 0.05 mM cobalt and with 0.1 mM cobalt) and NM522/p56 (without cobalt, with 0.05 mM cobalt and with 0.1 cobalt).
(2) in LB/2 : NM522 (negative control),PHL818 (positive control),NM522/piG3 (without cobalt and with 0.1mM cobalt),NM522/p10 (without cobalt and with 0.1mM cobalt),S19(without cobalt,with 0.1 mM cobalt and with 0.15 mM cobalt) and NM522/p56 (without cobalt, with 0.1 mM cobalt and with 0.15 cobalt).
Others
Start of 2X5 mL cultures of NMYO ( 5mL LB, 50µL saturated NMYO, 50µL Kan)
Because no NiCoT plasmid was correct, the transformation was abandonned.
