Team:Pasteur Paris/Microbiology week9

Aim: Check if the sillification is working with different volume of HCl and TEOS.

Protocol: follow in this link

What we did in the lab:
Materials:
• 1.5 ml Eppendorfs
• Silification protein
• HCl 1 M
• TEOS (Tetraethyl ortho silicate, Sigma)
• Shaking incubator
• 15 ml Falcon
• Buffer A (See protein purification protocol)
• 10 μl and 200 μl pipettes

Method:
1. In a 1.5 ml Eppendorf prepare a sample with the fraction that contains protein c2, add 1 ml of HCl 1mM, 65.8 μl of TEOS and let it incubate for 4 minutes in the rotating incubator 2. In a 15 ml Falcon, put 9 ml of buffer A and add the previous sample 3. Follow if silification initiates and progresses at various times :

Table 1
Times	Comments
10 minutes	Ø
15 minutes	Microscopic crystalline specs appear
30 minutes	Microscopic crystalline specs continue to appear
50 minutes	Microscopic crystalline specs continue to appears + crystalline specs grow and form a precipitate

4. Redo this experiment with 1 ml of TEOS, 50 μl of protein and vortex:

Table 2
Times	Comments
15 minutes	Crystalline specs appear
2 hours	Crystalline specs continue to appear

5. Redo the experiment with 1 ml of TEOS to see the catalytic activity of the protein :

Table 3
Times	Comments
1h45	Ø

6. Redo the experiment with 50 μl of HCl 1 mM and 50 μl of protein
7.Redo the experiment with 50 μl of HCl 1 mM but without our protein

Results:
When glycerol is added to the sample, as an amphiphilic modifier, an emulsion takes place which mean that glycerol act as a surfactant.
Our protein is a catalyst

Aim: Test the specificity of primers For and Rev to obtain more insert. We also switched to the Takara EX DNA polymerase for its terminal deoxynucleotide transferase, which adds an dA at the 3' end of the PCR product

Protocol: follow in this link

What we did in the lab:
Materials:
• dNTP mix
• MgCl₂ 25 mM
• EX polymerase buffer 10X
• RNAse free water
• C1⁄C2 insert DNA
• primers For and Rev
• Takara EX DNA polymerase
• PCR machine
• 10 μl and 200 μl pipettes

Method:
1. Use a Globalmix with :

Table 4
	Volumes (μl)
dNTP	25
Mgcl₂	25
Buffer 10X	50
RNAse free water	36.5
Final	50

2. For the next step use :

Table 5
Tube	Name	Premix (μl)	C1 (μl)	C2 (μl)	Primer For (μl)	Primer Rev (μl)
1	C1 insert (For +Rev)	46.5	1	Ø	1	1
2	C1 For	46.5	1	Ø	1	Ø
3	C1 Rev	46.5	1	Ø	Ø	1
4	C2 insert (For +Rev)	46.5	Ø	1	1	1
5	C2 For	46.5	Ø	1	1	Ø
6	C2 Rev	46.5	Ø	1	Ø	1
7	Without DNA	46.5	Ø	Ø	1	1
8	C1 without primer	46.5	1	Ø	Ø	Ø
9	C2 without primer	46.5	Ø	1	Ø	Ø

3. For the PCR program, start at 24°C, add 0.5 μl of Takara enzyme and proceed to 94°C

Aim: Verification of the content of the fractions obtained from the Ni-NTA column chromatography.

“”/ — Aim: Test the specificity of primers For and Rev to obtain more insert.

Protocol: follow in this link

What we did in the lab:
Materials:
• Loading buffer 6X
• PCR products
• Inserts C1 v2, C2 v2
• Primer S
• Primer AS
• Electrophoresis chamber, and power supply
• 10 μl pipette

Method:
1. Add 5 μl of DNA and 1 μl of buffer 6X to each sample 2. Use 6 μl of molecular weight marker and deposit each sample in the wells :

Table 6

L1 L2 L3 L4 L5 L6 L7 L8 L10 L11 L12

M. weight Marker Ø C1 + insert C1 + primer S C1 + primer AS C2 + insert C2 + primer S C2 + primer AS Without DNA C1 without primer C2 without primer

2. Start the electrophoresis at 50 V and 0.01 A for 10 minutes, then put it at 90 V and 0.03 A

Result
Figure 1

Aim: Get back the DNA amplified by PCR.

Aim: Increase the quantity of DNA for cloning.

Protocol: follow in this link

Results :

Figure 2

Aim: Test the specificity of primers For and Rev to obtain more insert.

Protocol: follow in this link

What we did in the lab:
Materials:
• Agarose
• Ethidium bromide drop
• Molecular weight ladder (Gene ruler 1 Kb, Thermofisher)
• Loading buffer 6X
• PCR products
• Electrophoresis chamber, power supply
• 10 μl pipette

Method:
1. Make an agarose gel at 0.7% 2. Prepare the samples with 5 μl of PCR products and 1 μl of loading buffer 6X 3. Depose each samples in the wells :

Table 7
L1	L2	L3	L4	L5	L6	L7	L8	L9	L10	L11	L12	L13	L14	L15
Ladder	Ø	A1	A2	Ø	B1	B2	Ø	D1	Ø	D2	Ø	E1	E2	Ø

4. Begin the electrophoresis at 50 V for 15 minutes, then at 90 V for 1h30

Result
There are streaks in lanes 3, 4, 9 and 11 which mean that the PCR did not work. But it seems to work for lanes 6, 7, 13 and 14.
The PCR must be done another time for A1⁄A2 and D11⁄D2 with a lower temperature (1°C).

Aim: Test the specificity of primers For and Rev to obtain more insert.

What we did in the lab:
Materials:
• Qiagen PCR clean up kit • PCR products • 200 μl pipette

Method:
1. Prepare the 8 samples:

Table 8
Samples	A1	A2	B1	B2	D1	D2	E1	E2
Buffer A (μl) <	90	90	90	90	90	90	90	90

2. Follow the protocol of the Qiagen PCR clean up kit

Result
We obtained 25 μl of each insert purified by PCR.

Aim: Test the specificity of primers For and Rev to obtain more insert.

Protocol: follow in this link

What we did in the lab:
Materials:
• PCR products
• Salt solution
• pET43.1 vector DNA
• Incubator 37°C
• 10 µl pipettes
• 1 ml Eppendorfs

Method:
1 For each inserts, prepare the following mix :

Table 9
	Volumes (μl)
PCR products	4
Salt solution	1
pET 43.1	1
Total	6

2. Let ligate for 5 minutes at room temperature 3. Incubate for 10 minutes at 65°C.

Aim: Check if our plasmid was inserted in the bacteria, and to recover larger amounts of it.

Protocol: follow in this link

Aim: Increase the quality of DNA.

Protocol: follow in this link

What we did in the lab:
Materials:
• dNTP
• MgCl₂
• Buffer 20X
• RNAse free
• 10 μl , 20 µl and 200 μl pipettes
• 1.5 ml Eppendorfs
• A1⁄A2 and B1⁄B2 inserts
• PCR machine
• 10 μl 20 µl and 200 μl pipettes
• 1.5 ml Eppendorfs

Method:
1. Prepare a Globalmix with :

Table 10
	Volumes (μl)
dNTP	12.5
MgCl₂	12.5
Buffer 20X	25
RNAse free	182.5
Primer S (For)	5
Primer AS (Rev)	5
Total	242.5

2. Prepare the following samples :

Table 11
	A1	A2	B1	B2
V_inserts (μl)	1	1	1	1
V_globalmix	48.5	48.5	48.5	48.5

3. Launch the PCR

Aim: Check the efficiency of the PCR.

Protocol: follow in this link

What we did in the lab:
Materials:
• Agarose
• Ethidium bromide drops
• Molecular weight ladder (Gene ruler 1 Kb, Thermofisher)
• Loading buffer 6X
• PCR products
• 10 μl pipette
• Electrophoresis machine
• 1.5 ml Eppendorfs

Method:
1. Make an agarose gel at 0.7% 2. Prepare the samples with 5 μl of PCR products and 1 μl of loading buffer 6X
3. Deposit each sample in the wells :

Table 12
L1	L2	L3	L4	L5	L6	L7	L8
Ladder (6 μl)	Ø	A1 : 5 μl of DNA + 1 μl of buffer 6X	A2 : 5 μl of DNA + 1 μl of buffer 6X	Ø	D1 : 5 μl of DNA + 1 μl of buffer 6X	D2 : 5 μl of DNA + 1 μl of buffer 6X	Ø

4. Begin the electrophoresis at 50 V for 15 minutes, then at 90 V for 1h30.

Aim: Get DNA back.

Protocol: follow in this link

Results

Figure 3

Aim: Make the inserts fitted with the plasmid.

Protocol: follow in this link

What we did in the lab:
Materials:
• Hind III (NEB)
• Xba I 5NEB)
• Buffer CutSmart 10X (NEB)
• H₂O
• 1.5 Eppendorfs
• C1 v2 and C2 v2
• Incubator 37°C
• 20 μl and 200 μl pipettes

Method:
1. Prepare a Globalmix :

Table 13
	Volumes (μl)
HindIII	20
XbaI	20
Buffer CutSmart 10X	50
H₂O	10
Total	100

2. Put 5 μl of the mix in each tube and add 20 μl of DNA C1 v2 and C2 v2
3. Let digest for 2 hours at 37°C and inactivate enzymes for 5 minutes at 65°C

Aim: Check if our inserts have been correctly digested by the enzyme.

Protocol: follow in this link

What we did in the lab:
Method:
Lane 1 :

Table 14
1	2	3	4	5	6	7	8	9	10	11	12
Ladder	Ø	C1.1		Ø	C1.2		Ø	C1.3		Ø	C1.4
14	15	16	17	18	19	20	21	22	23	24	25
Ø	C1.5		Ø	C1.6		Ø	C1.7		Ø	C1.8

Lane 2:

Table 15
1	2	3	4	5	6	7	8	9	10	11	12
Ladder	Ø	C2.2		Ø	C2.3		Ø	C2.4		Ø	C2.5
14	15	16	17	18	19	20	21	22	23	24	25
Ø	C2.6		Ø	C2.8		Ø	C2.9		Ø	Ø	Ø

Aim: Get back the DNA which was digested and purified.

Protocol: follow in this link

What we did in the lab:
Materials
• Qiagen Gel Extraction Kit

Method:
Use Qiagen extraction kit with a final volume of 50 μl.

Results

Table 16
Sample	Weight of gel (g)
C1.2	1.3435
C1.4	1.3564
C1.5	1.3825
C1.6	1.4318
C1.7	1.4392
C1.8	1.3564
C1.10	1.3800
C2.1	1.3179
C2.2	1.2500
C2.3	1.3910
C2.9	1.3668
C2.10	1.3934

Aim: Put the insert into the plasmid and transform the plasmid into bacteria.

Protocol: follow in this link

Results (on the August 4, 2016)
Several colonies have grown but they are too small so we let them grow more. But C1.2 has much more colonies so we spread it into another Petri dish.

Aim: Get back the DNA from bacteria.

Protocol: follow in this link

Results

Figure 4 Figure 5

Aim: Prepare the ligation.

Protocol: follow in this link

What we did in the lab:
Materials
• 1.5 ml Eppendorfs
• DNA
• Buffer CutSmart (NEB)
• HindIII (NEB)
• XbaI (NEB)
• H₂O
• Samples B1⁄B2, E1⁄E2
• 10 μl and 20 μl pipettes

Method:
1. Make a master mix with :

Table 17
Volumes (μl)	pET 43.1	B1⁄B2⁄E1⁄E2
DNA	7.5	7.5
Buffer CutSmart	2.5	7.5
HindIII	1	1
XbaI	1	1
H₂O	1.5	0.5
Total	25	25

2. Distribute the volumes in each tube

Aim: Avoid the religation of the plasmid with itself.

Protocol: follow in this link

What we did in the lab:
Materials
• 1.5 ml Eppendorfs
• Digested DNA
• Dephosphorylase rSAP (recombinant Shrimp alkaline phosphatase) (NEB)
• Buffer CutSmart
• H₂O
• Incubator 37°C
• 10 μl and 20 μl pipettes

Method:
1. In a 1.5 ml Eppendorf, put :

Table 18
	Volumes (μl)
Digested DNA	9
Dephosphorylase	0.2
Buffer CutSmart	2
H₂O	8.8
Total	20

2. Incubate one hour at 37°C
3. Incubate 5 minutes at 65°C

Aim: Increase the quantity of DNA.

Protocol: follow in this link

What we did in the lab:
Materials
• dNTP mix
• MgCl₂ 25 mM
• Buffer 10X
• RNAse free water
• Primer S
• Primer AS
• Samples A1⁄A2 and D1⁄D2
• 10 μl , 20 μl and 200 μl pipettes
• PCR machine
• Takara DNA polymerase enzyme
• 1.5 ml Eppendorfs

Method:
1. Prepare the mix :

Table 19

Volumes (μl)

**dNTP**
12.5

**MgCl₂**
Ø

**Buffer 10X**
25

**RNAse free**
195

**Primer S**
5

**Primer AS**
5

**Total**
242.5

2. To put the sample into the PCR machine, use the table :

Table 20

Tube Names Mix (μl) A1 (μl) A2 (μl) D1 (μl) D2 (μl)

1
A1 48.5 1 Ø Ø Ø

2
A2 48.5 Ø 1 Ø Ø

3
D1 48.5 Ø Ø 1 Ø

4
A1 48.5 Ø Ø Ø 1

3. For hot start PCR, launch the PCR machine and once it is at 95°C, add 0.5 μl of Takara enzyme in each tube

Results
Figure 6

Aim: Have more bacteria with the insert A.

Protocol: follow in this link

What we did in the lab:
Materials
• 50 ml Falcon
• LB medium
• 20 ml and 20 μl pipettes
• TOPO
• C1 v2 and C2 v2
• Carbenicillin 50 mg⁄ml
• Incubator
• LB medium

Method:
1. In a 50 ml Falcon, put 15 ml of LB medium
2. Add 15 μl of carbenicillin
3. Add a colony in the Falcon. But for C1 v2 (2) we had to wait one more night because bacteria had not grown enough
4. Let it incubate at 37°C

Aim: Check if the PCR is efficient.

Protocol: follow in this link

What we did in the lab:
Materials
• Agarose
• A1⁄A2 and D1⁄D2
• Molecular weight marker ladder (Gene ruler 1 Kb, Thermofisher)
• Loading buffer 6X
• 10 μl pipette

Method:
1. Make a 0.7% agarose gel
2. Add 5 μl of DNA and 1 μl of loading buffer. But we used 10 μl of DNA for the sample 4
3. Depose the sample for the electrophorese like this :

Table 21
L1	L2	L3	L4	L5	L6	L7	L8	L19
Ladder 6 μl	Ø	(1)	Ø	(2)	Ø	(3)	Ø	(4)

Results
A lot of streaks appeared so it did not work as expected. We have to redo this experiment with the gradient mode

Aim: Increase the quantity of DNA.

Protocol: follow in this link

What we did in the lab:
Materials
• dNTP mix
• MgCl₂ 25 mM
• Buffer 10X
• RNAse free
• Primer S
• Primer AS
• A1⁄A2 and D1⁄D2
• 200 μl pipette

Method:
1. Make a Globalmix with :

Table 22

Volumes (μl)

**dNTP**
72.5

**MgCl₂**
Ø

**Buffer 10X**
145

**RNAse free**
1131

**Primer S**
29

**Primer AS**
29

**Total**
1206.5

2. Prepare the samples :

Table 23

Name Samples Globalmix (μl) DNA (μl)

A1
to (7) 48.5 1 of A1

A2
(8) to (14) 48.5 1 of A2

D1
(15) to (21) 48.5 1 of D1

D2
(22) to (28) 48.5 1 of D2

3. Deposit the samples on the gel:

Table 24

A1 A2 D1 D2

A
(1) (8) (15) (22)

B
(2) (9) (16) (23)

C
(3) (10) (17) (24)

D
(4) (11) (18) (25)

E
(5) (12) (19) (26)

F
(6) (13) (20) (27)

G
(7) (14) (21) (28)

4. Make a 0.7% agarose gel and perform an electrophoresis with 5 µl of DNA and 1 µl of loading 6X buffer for each sample

Results
Figure 7

Aim: Increase the DNA.

What we did in the lab:
Materials
• C1⁄C2
• Na Acetate (NaOAc)
• Ethanol (70%)
• Incubator
• Buffer E
• 200 μl and 1000 μl pipette

Method:
1. Calculate the final volume for each insert :
For C1 : 50×7 = 350 μl
For C2 : 50×5 = 250 μl
2. Add 1⁄10 of the volume of NaOAc and 2.5x volume of ethanol :
For C1 : 35 μl of NaOAc and 875 μl of ethanol
For C2 : 25 μl of NaOAc and 625 μl of ethanol
3. Mix the samples and incubate 8minutes at -80°C
4. Centrifuge 5 minutes at 4°C and 15000 RPM. The, throw out the supernatant
5. Add 1 ml of ethanol ice cold (70%)
6. Centrifuge 15 minutes at 4°C and 15000 RPM
7. Throw out the supernatant and let dry at room temperature
8. Resuspend the pellet in 50 μl of buffer E.

Results

Figure 8

Aim: Make the inserts fitted with the plasmid.

Protocol: follow in this link

What we did in the lab:
Materials
• 10 μl, 20 μl and 200 μl pipette
• Hind III (NEB)
• XbaI (NEB)
• Buffer CutSmart
• H₂O
• B1⁄B2 and C1⁄C2

Method:
1. Make a Globalmix with :

Table 25
	Volumes (μl)
HindIII	45
XbaI	45
Buffer CutSmart	112.5
H₂O	67.5
Total	270

2. Distribute this global mix in each of our 4 tubes so they will contain :

Table 26
	Volumes (μl)
DNA	20
HindIII	1
XbaI	1
Buffer CutSmart	2.5
H₂O	1.5
Total	26

3. Follow the protocol for digestion.

Aim: Prepare the plasmid for the transformation.

Protocol: follow in this link

What we did in the lab:
Materials
• C1⁄C2
• pET43.1
• T4 ligase
• Buffer 10X
• H₂O
• Incubator
• 10 μl and 20 μl pipettes

Method:
1. In a 1.5 ml eppendorf, put :

Table 27
	C1	C2	pET 43.1 (a+) alone
C1 (μl)	15	Ø	Ø
C2 (μl)	Ø	15	Ø
pET 43.1 (μl)	4	4	4
T4ligase (μl)	1	1	1
Buffer 10X (μl)	2.2	2.2	2.2
H₂O (μl)	Ø	Ø	15
Total (μl)	22.2	22.2	22.2

2. Incubate for 1 hour at room temperature 3. Incubate for 10 minutes at 65°C

Aim: Express our plasmid in bacteria.

Protocol: follow in this link

What we did in the lab:
Materials
• Qiagen Extraction Kit

Method:
Use the Qiagen gel extraction kit for a final volume of 50 μl.

Table 28
Colonies	Δm (mg)
B1 colony 1	136
B1 colony 2	170
B2 colony 4	166
B2 colony 7	175
E1 colony 1	110
E1 colony 2	143
E2 colony 1	108
E2 colony 2	128

Aim: Ligate our inserts with the plasmid.

Protocol: follow in this link

What we did in the lab:
Method:
We use the next volumes:

	Volumes (μl)
Insert (B1 or B2 or C1 or C2)	15
pET43.1 (100 ng)	2
T4 ligase	1
Buffer 10X	2
Total	20

Aim: Put our ligated plasmid into bacteria.

Protocol: follow in this link

Results
48 hours later, no colony had grown so the transformation did not work, it has to be redone.

120. Silification tests

121. PCR of C1 v2 and C2 v2 with Takara enzyme

122. Agarose gel

123. Electrophoresis of the PCR products (C1 v2 and C2 v2)

124. PCR Clean up

August 2, 2016:

125. PCR of A1, A2, B1, B2, D1, D2, E1 and C2

126. Analysis of PCR products from August 1

127. PCR clean up

128. Ligation of PCR products with TOPO cloning

129. Transformation in TOP10 competent cells

130. PCR of A1⁄A2 and D1⁄D2

August 3, 2016:

131. Analysis of PCR products from August 2, 2016

132. Miniprep of cultures C1 v2 and C2 v2 from August 2

133. Digestion of C1 v2 and C2 v2

134. Electrophoresis of C1 v2 and C2 v2

135. DNA extraction from the gel

136. Ligation of C1.2⁄C1.5 and C2.1⁄C2.2 with pET43.1 (a+) and transformation with TOP10 competent cells

August 4, 2016:

137. Miniprep of B1⁄B2 and E1⁄E2

138. Digestion of B1⁄B2, E1⁄E2 and pET43.1 (a+) with Hind III and Xba I

139. Dephosphorylation of digested pET43.1 (a+)

140. PCR of A1⁄A2 and D1⁄D2 without MgCl₂

141. Preculture of TOPO – C1 v2 (2) and (5) and TOPO – C2 v2 (1) and (2)

142. Electrophoresis of PCR products A1⁄A2 and D1⁄D2

143. Next PCR of A1⁄A2 and D1⁄D2

144. Pool of inserts C1 v2 and C2 v2

August 5, 2016:

145. Digestion of B1⁄B2 and C1⁄C2

146. Ligation of C1 and C2 with pET43.1 (a+) digested and dephosphorylated

147. Transformation of TOP10 competent cells

148. Ligation of B1⁄B2⁄C1⁄C2 in pET43.1 (a+)

149. Transformation of B1⁄B2⁄C1⁄C2 in TOP10

Team:Pasteur Paris/Microbiology week9

click on the weeks

Week 9

August 1, 2016:

	Volumes (μl)
dNTP	72.5
MgCl₂	Ø
Buffer 10X	145
RNAse free	1131
Primer S	29
Primer AS	29
Total	1206.5

Name	Samples	Globalmix (μl)	DNA (μl)
A1	to (7)	48.5	1 of A1
A2	(8) to (14)	48.5	1 of A2
D1	(15) to (21)	48.5	1 of D1
D2	(22) to (28)	48.5	1 of D2

	A1	A2	D1	D2
A	(1)	(8)	(15)	(22)
B	(2)	(9)	(16)	(23)
C	(3)	(10)	(17)	(24)
D	(4)	(11)	(18)	(25)
E	(5)	(12)	(19)	(26)
F	(6)	(13)	(20)	(27)
G	(7)	(14)	(21)	(28)

Team:Pasteur Paris/Microbiology week9

click on the weeks

Week 9

August 1, 2016:

120. Silification tests

121. PCR of C1 v2 and C2 v2 with Takara enzyme

122. Agarose gel

123. Electrophoresis of the PCR products (C1 v2 and C2 v2)

124. PCR Clean up

August 2, 2016:

125. PCR of A1, A2, B1, B2, D1, D2, E1 and C2

126. Analysis of PCR products from August 1

127. PCR clean up

128. Ligation of PCR products with TOPO cloning

129. Transformation in TOP10 competent cells

130. PCR of A1⁄A2 and D1⁄D2

August 3, 2016:

131. Analysis of PCR products from August 2, 2016

132. Miniprep of cultures C1 v2 and C2 v2 from August 2

133. Digestion of C1 v2 and C2 v2

134. Electrophoresis of C1 v2 and C2 v2

135. DNA extraction from the gel

136. Ligation of C1.2⁄C1.5 and C2.1⁄C2.2 with pET43.1 (a+) and transformation with TOP10 competent cells

August 4, 2016:

137. Miniprep of B1⁄B2 and E1⁄E2

138. Digestion of B1⁄B2, E1⁄E2 and pET43.1 (a+) with Hind III and Xba I

139. Dephosphorylation of digested pET43.1 (a+)

140. PCR of A1⁄A2 and D1⁄D2 without MgCl2

141. Preculture of TOPO – C1 v2 (2) and (5) and TOPO – C2 v2 (1) and (2)

142. Electrophoresis of PCR products A1⁄A2 and D1⁄D2

143. Next PCR of A1⁄A2 and D1⁄D2

144. Pool of inserts C1 v2 and C2 v2

August 5, 2016:

145. Digestion of B1⁄B2 and C1⁄C2

146. Ligation of C1 and C2 with pET43.1 (a+) digested and dephosphorylated

147. Transformation of TOP10 competent cells

148. Ligation of B1⁄B2⁄C1⁄C2 in pET43.1 (a+)

149. Transformation of B1⁄B2⁄C1⁄C2 in TOP10

140. PCR of A1⁄A2 and D1⁄D2 without MgCl₂