Difference between revisions of "Team:Pasteur Paris/Microbiology week6"

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 600 nm (Abs_{600 nm}) to make a growth curve (Ultrospec 3100 pro, GE Lifesciences) using plain plastic cuvettes, 1 cm path length.

Time	C2 1.1 Abs_{600 nm}	C2 1.2 Abs_{600 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

Table 35

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: OD_{600 nm} C2 1.1= 0.865, OD_{600 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.

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:Integrate the inserts into the plasmid

Protocol:follow in this link

Aim:Purify the digested inserts (C1/C2/B1/B2) and the plasmid before the ligation.

Protocol: follow in this link

What we did in the lab:
Method:
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

Aim:To Prepare 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

Table

Aim:Transform our plasmids into competent cells to amplify the DNA.

Protocol: follow in this link

What we did in the lab:
Method:
Moreover, we spread the competent cells from July 7,2016 on petri dishes to have new colonies.

Aim: As our culture of BL21DE3 didn’t work we redid exactly the same experiment as yesterday.

	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

Table 38

Aim:Check if the bacteria that were induced by IPTG have produced the protein (weight = 30 kDa).

What we did in the lab:
Materials:
• mini protean TGX 4-15% precast gels (BioRad)
• TGS Buffer 10X
• Electrophoresis chamber
• 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 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.

Protocol: follow in this link

What we did in the lab:
Method:
Once, the DNA has been pelleted let the Eppendorf open to evacuate the ethanol (all the week-end).

@@ Line 342: / Line 342: @@
                  <U>Method:</U></br>
                  In a 1 ml Eppendorf, we put:   </br>
-                     - 25 &#181;l of pET43.1 (3 &#181g/50 &#181l)  </br>
+                     - 25 &#181;l of pET43.1 (3 &#181;g/50 &#181;l)  </br>
                      - 2 &#181;l of buffer Cutsmart 10X  </br>
                      - 1 &#181;l of SAP  </br>

Difference between revisions of "Team:Pasteur Paris/Microbiology week6"

Revision as of 02:57, 19 October 2016

click on the weeks

Microbiology Notebook

Week 6

July 11, 2016:

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

July 12, 2016:

80. Results of the culture of BL21DE3 (July 11, 2016)

July 13, 2016:

81. Protein gel

82. Extraction of pET43.1a(+)