74. Culture of BL21DE3
75. Dephosphorylation of pET43.1a(+)
76. Digestion of the new inserts
77. Electrophoresis on agarose gel
78. Ligation of the inserts
79. Transformation of competent cells
80. Results of the culture of BL21DE3 (July 11, 2016)
81. Protein gel
82. Extraction of pET43.1a(+)
Aim: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. Protocol: follow in this link What we did in the lab: Materials: • 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 600nm to make a growth curve (utrospec 3100 pro) using plain platic cuvettes, 1 cm path length.
| Time | C2 1.1 Abs600nm | C2 1.2 Abs600nm |
|---|---|---|
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 | 50,675 |
Aim:after the digestion we dephosphorylate It to prevent that pET43.1 binds without the insert. Protocol: follow in this link What we did in the lab: Method: In a 1 ml Eppendorf, we put: - 25 µl of pET43.1 (3 µg/50 µl) - 2 µl of buffer Cutsmart 10X - 1 µl of SAP TOTAL = 28 µl
Aim:Purify the inserts digested (C1/C2/B1/B2) and the plasmid before the ligation. Protocol: follow in this link What we did in the lab: Method: 1. make an 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
Aim:TPrepare our plasmids for the transformation. Protocol: follow in this link What we did in the lab: Method: For each insert, we make two samples.
| 1:1 | 1:3 | |
|---|---|---|
DNA (200 ng) |
4 µl | 4 µl |
Insert B1/B2/C1/C2 (version 2) |
4 µl | 12 µl |
T4 ligase |
1 µl | 1 µl |
Buffer 10x |
2 µl | 2 µl |
H20 |
9 µl | 1 µl |
TOTAL |
20 µl | 20 µl |
Aim:put our plasmids in competent cells to have multiplication of our plasmid. Protocol: follow in this link What we did in the lab: Method: Moreover, we spread the competent cells from July 7,2016 on petri dish to have new colonies.
Aim:Check in the bacteria induced by IPTG have producted the protein (weight = 30kDa). Protocol: follow in this link What we did in the lab: Materials: • mini protean TGX 4-15% precast gels (bioRad) • TG5 Buffer 10X • Cuve • Samples of our culture C2 1.1 (-) IPTG C2 1.1 (+) IPTG C2 1.2 (-) IPTG C2 1.2 (+) IPTG Method: 1. Put the gel in the cuve, take off the green parts 2. Fill the cuve with the buffer 3. Follow the deposit table: L1: 8 µl ladder protein Thermofisher 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 denaturate 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:get back the plasmid fro the cultures made on the July 11, 2016. Protocol: follow in this link What we did in the lab: Method: Once, the DNA is pelleted let the Eppendorf open to evacuate ethanol (all the week-end).
