Make a growth curve and induce the production of protein. We will start producing our protein in bacterial cells. In order to do that we need to have an idea of the growth profile of the cells with this construct inside. This will determine at what time we can induce the expression of our protein.
• Erlenmeyer flasks 250 ml capacity
• IPTG (Iso-propyl β thio-galactopyranoside) 0.5 M
• LB media, carbenicillin at 50 mg/ml
• Shaking incubator (Infors HT)
• Spectrophotometer.
Method:
1. 2 x 250 ml Erlenmeyer, put 25 ml of LB and 25 µl of carbenicillin at 50 mg/ml
2. then add in one colony of BL21DE3 pLys S with C2 1.2 and in the other flask add one with C 1.1
3. put them 1 hour at 37°C and 180 RPM
4. Store the master plate (reference) at -4°C and make a copy plate grown at 37°C, then stored at 4°C.
5. Measure absorbance at 600 nm (Abs600 nm) to make a growth curve (Ultrospec 3100 pro, GE Lifesciences) using plain plastic cuvettes, 1 cm path length.
Table 52
Time
C2 1.1 Abs600 nm
C2 1.2 Abs600 nm
13h59
0.128
0.044
14h25
0.168
0.095
14h50
0.282
0.188
15h17
0.387
0.262
15h32
0.722
0.620
16h07
0.675
1. When absorbance reached 0.7, we added IPTG at 0.5 mM to induce the production of protein
For C2 1.1 we added IPTG at 16h
and for C2 1.2 we added IPTG at 16h09
2. We let the induction proceed during 3 hours at 37°C and 180 RPM
Measure of absorbance: OD600 nm C2 1.1= 0.865, OD600 nm C2 1.2 =0.846
Results:
Our experiment failed because the agitation was stopped ! (Someone unfortunately either didn't restart the incubator shaking mode or programmed a small time on the shaker's timer by error). We pelleted 1 ml of each culture (3 min at 6800 g) and stored at -20°C. The Erlenmeyer content was pelleted too (5 min at 4500 RPM) and stored at -80°C.
In the meanwhile, during the growth curve experiment, we performed all the steps for the transformation of the new versions of our inserts synthesized by IDT.
We indeed decided to re-synthesized our inserts, such that it had a few improvements, namely that now it'll depend on the vector's promoter, the linker regions had less GC content, and were shortened. We will refer to these constructs as X.V2.
Aim:
After the digestion we dephosphorylated it to prevent that pET43.1 to religate without the insert.
1. make a 0.7% agarose gel
2. do an electrophoresis following the deposit table
L1: ladder (Gene Ruler)
L3: B1
L5: B2
L7: C1
L9: C2
3. we extract the DNA purified o the gel with QIAGEN extraction kit. We follow the kit step
Gel mass: B1: 304 mg B2: 382 mg C1: 378 mg C2: 451 mg
1. Put the gel in the electrophoresis chamber, take off the green parts off the gel before
2. Fill the chamber with the buffer 1X
3. Follow the deposit table:
L1: 8 µl ladder protein Thermofisher (PAGE Ruler plus)
L3: 17 µl of C2 1.1 (-) IPTG
L6: 17 µl of C2 1.1 (+) IPTG
L8: 17 µl of C2 1.2 (-) IPTG
L10: 17 µl C2 1.2 (+) IPTG
In each eppendorg we added:
- 10 µl of protein solution
- 10 µl of laemli 2X
Heat the samples at 95°C for 5 min to denature the protein
4. start of migration at 130 V
5. Stop the migration about one hour later
6. Open the plastic content to get back the gel
7. Wash three times five minutes in distilled H20
8. Fill a cuve with Bleu de Coomassie and let color the gel during 30 minutes. Let one hour more if required
9. Wash distilled H20 until the bands appear
Results:
It seems to have a slight band for C2 11 (+) IPTG that could be our protein but this band doesn’t appear in C2 1.2 (+) IPTG. We decided to let it the whole weekend in distilled H20 to analyze the bands.
Aim:
To get back the plasmid from the cultures made on the July 11, 2016.
