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Team:Aix-Marseille/Notebook 07
From 2014.igem.org
Notebook: July
Week 1 : 06/30/2014 ➡ 07/06/2014 Preparation of strains
- W3110 Escherichia coli strain and DH5alpha bacteria were put in culture on Petri dish.
- Both strains were incubated in Erlenmeyer flasks at 37 ° C.
- Preparation to electrocompetence : W3110 strain was washed in a 10% glycerol solution to remove extracellular salts that are lethal in this processing. W3110 competent bacteria were then frozen and stored.
- Preparation to chemocompetence : working at cold temperature, bacteria were washed and resuspended with CaCl2. Again, bacteria were washed and resuspended with CaCl2 and glycerol. Cells were frozen at -80°C.
- Electrocompetence : W3110 bacteria were transformed with pKOBEG plasmid by electroporation and put in culture at 30°C on petri dish.
- Colonies were observed in W3110 control cultures. Transformed bacteria (W3110pKOBEG) were put in culture at 30°C.
- Chemocompetence : A heat shock at 37°C was made with DH5α strain and the bacteria were then transformed with pBba_JO4450 plasmid and put in culture on petri dish.
- W3110pKOBEG bacteria were washed and arabinose was added to provide the expression of the recombinase encoded by pKOBEG plasmid. They were then incubated at 42 °C in order to destroy the thermosensitive pKOBEG plasmid.
- Preparation of electrocompetence : W3110 bacteria expressing the recombinase were prepared to electrocompetence and frozen at -80 ° C.
No colony was observed in the petri dish where DH5α transformed bacteria were previously put in culture. - Chemocompetence : bacteria were prepared using a formerly thawed aliquot. A heat shock at 42°C were made on competent DH5α containing pBba-J04450. They were finally put in culture at 37°C.
- DH5α control : Colonies were observed in the “negative control” culture. These colonies did not express RFP (no red fluorescent colour could be observed).
The LB we used in our experiments were contaminated, leading to the growth of undesirable colonies. The transformation of W3110 using pKOBEG plasmid was succesfull. The recombinase was expressed and the pKOBEG plasmid was destroyed using a thermosensitive origin of replication.
Week 2 : 07/07/2014 ➡ 07/13/2014
- Supercompetent DH5alpha preculture was made.
- Electroporation of W3110 strain with J04450 plasmid (20ng) and the telltale without plasmid was made.
- Preparation of supercompetent cells
The plasmid concentration contained in the iGEM kit was enough to transform bacteria by electroporation.
Week 3 : 07/14/2014 ➡ 07/20/2014
- DH5α supercompetent bacteria were transformed using 3 ng of J04450 plasmid or 60 pg of J04450 plasmid. Both cultures showed colonies meaning transformations were successful.
- DH5α supercompetent bacteria were transformed with respectively 1,5 ng of the following plasmids :
- K864600 plasmid
- B0032 plasmid
- B0033 plasmid
- E1010 plasmid
- B0034 plasmid
- B0010 plasmid
- B0030 plasmid
- E0040 plasmid
- pT18 RelA
- pBAD Mesh1
- DH5α bacteria containing respectively the following plasmids were put in liquid culture in order to perform miniprep the day after.
- K864600 plasmid
- B0032 plasmid
- B0033 plasmid
- E1010 plasmid
- B0034 plasmid
- B0010 plasmid
- B0030 plasmid
- E0040 plasmid
- DH5α supercompetent bacteria were transformed with respectively :
- pSB1C3 K608006 Cm resistant plasmid
- pSB1C3 K823017 Cm resistant plasmid
- pSB1C3 J04500 Cm resistant plasmid
- pWW2179 (SH3)4 Cm resistant plasmid
- pWW2021 CusR-L-LZa Kan resistant plasmid
- pWW2181 Amp resistant plasmid
- As a negative control, a culture of empty bacteria and Ampicillin was made.
- After the Miniprep was performed on bacteria containing pSB1AC3-J04450 plasmid, an electrophoresis was realised. It shows one high molecular weight band. The plasmid pSB1AC3-J04450 was successfully purified. It is stocked at -20°C and the strain is conserved in glycerol at -80°C.
Note :
Petri dishes containing respectively bacteria transformed with pT18 RelA plasmid and bacteria transformed with pBAD Mesh1 plasmid showed so many colonies that it was not possible to transfer isolated colonies into liquid culture. So, new cultures of both transformed bacteria were made again using a technique leading to isolated colonies.- After the Miniprep was performed on bacteria containing K864600 plasmid to E0040 plasmid, the following electrophoresis* was obtained :
* Be aware that Well 1 was filled with 5μL whereas the others were filled with only 3μL.
- Well 1: B0010
- Well 2: B0030
- Well 3: B0032
- Well 4: B0033
- Well 5: B0034
- Well 6: E0040
- Well 7: E1010
- Well 8: K864600
- Petri dish containing the bacteria transformed the day before with p5B1C3 K608006 Cm resistant plasmid to DH5α
- DH5α supercompetent bacteria previously transformed ( on 07/17/2014) with pSB1C3 K608006 Cm resistant plasmid to p5B1C3 K608006 Cm resistant plasmid were put in liquid culture in order to perform miniprep the day after
Week 4 : 07/21/2014 ➡ 07/27/2014
- Several plasmids were purified thanks to Macherey Nagel MiniPrep Kit :
- pSB1C3 K608006
- pSB1C3 K823017
- pSB1C3 J04500
- pWW2179 (SH3)4 Cm resistant
- pWW2021 CusR-L-LZa Kan resistant
- pWW2181 Amp resistant
- pT18 RelA Amp resistant
- pBAD Mesh1 Amp resistant
Each well contains one or two fragments of DNA (corresponding to the different plasmid compaction degrees), thus all plasmids were successfully purified. They were stocked at -20°C and strains were put in glycerol at -80°C. - Bacteria transformed with the pSB1A2 BBa_B0010 plasmid were strangely red. That's why the plasmid was twice digested with EcoR1 and with XBa1 and Pst1. Digestion products were tested thanks to agarose gel electrophoresis. Results are presented below.
The digestion with EcoR1 made the plasmid linear. The digestion with Xba1 and Pst1 was supposed to produce two bands, corresponding to the backbone (2070pb) and to the BBa_B0010 part (80pb). No low molecular weight band appeared, the only visible band has a too low molecular weight to be the expected backbone. Something is wrong. We have to clarify this. Further experiments should be made to clarify these surprising results.
- The pSB1A2 BBa_B0010 plasmid was anew twice digested with EcoR1 and with XBa1 and Pst1. Digestion products are tested thanks to agarose gel electrophoresis. Results are presented below.
Again, these results did not make sense. It looks as if restriction enzymes did not cut the plasmid. This part is set-aside for the moment.
- pSB1C3 BBa_E1010, pSB1A2 BBa_E0040 and pSB1A2 BBa_B0034 plasmids were digested and ligated according to the BioBrick Assembly Kit of BioLabs. The destination plasmid DNA used is the backbone pSB1K3.m1 linearized plasmid. DH5α supercompetent bacteria were transformed with ligation products and spread over Kan dish
- DH5α supercompetent bacteria were transformed with BBA_J23100 (17D Plate 4) and spread over Amp dish.
- DH5α supercompetent bacteria previously transformed (on 23/17/2014) with BBA_J23100 Amp resistant plasmid and with Brick 200 and 202 Kan resistant plasmid were put in liquid culture in order to perform miniprep the day after
- Several plasmids were purified thanks to Macherey Nagel MiniPrep Kit :
- Brick 200 (1)
- Brick 200 (2)
- Brick 202 (1)
- Brick 202 (2)
- J23100
Each well contains DNA fragments, thus all plasmids were successfully purified. - Bricks 200 (1), 200 (2), 202 (1) and 202 (2) were digested by EcoR1 and Spe1. Digestion products are tested thanks to agarose gel electrophoresis. Results are presented below.
To control it was the correct insert, a second electrophoresis was made with the RFP and GFP digestion products. Results are presented below.
Brick 200 (1), 200 (2) and 202 (1) did not present any insert. Only Brick 200 (2) seemed to present an insert.
Results were not conclusive. A control PCR might be made in order to determine the presence or the absence of BBa_B0034 RBS.
