Team:Pasteur Paris/Microbiology week10

Aim: Increase the quantity of DNA before extraction.

Protocol: follow in this link

What we did in the lab:
Materials:
• 50 ml Falcon tube
• Shaking incubator (INFORS HT)
• Swing bucket centrifuge (JOUAN GR41)
• Colonies of C2 v2 and B1 v2
• Carbenicillin at 50 mg⁄ml
• LB medium
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)

Method:
1. In a 50 mL falcon, put 48 ml of LB and 48 μl of carbenicillin.
2. For B1 v2 :
2.a Prepare 13 eppendorfs of 1.5 ml in which add 1 ml of the previous mix.
2.b Take with a toothpick colonies on the petri dish.
2.c Place the toothpick in a tube.
2.d Let incubate overnight at 37 °C and 150 rpm.
3. For C2 v2 :
3.a Prepare 20 eppendorfs of 1.5 m with 1 m of LB and carbenicillin mix.
3.b Take colonies and place it as previously explained.
3.c Let incubate overnight.

Aim: As the transformations did not work (B2, E1 and E2 in pET 43.1 (a+) ) with TOP 10 competent cells, we take the products of the midiprep done on the 4^th of August and we digest before redoing the transformation.

Protocol: follow in this link

What we did in the lab:
Materials:
• Restriction enzymes: Xba I, Hind III (New England Biolabs, NEB)
• Restriction enzyme buffers
• 37 &176;C water bath
• Shaking incubator (INFORS HT)
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)

Method:
1. Realize a Mastermix and store it on ice :

Table 1
Reactants	Volumes (µl)
XbaI	30
HindIII	30
Buffer 2.1	90
Distilled water	90
Total	150

2. In For each insert (B2, E1, E2), prepare 10 tubes of 1.5 ml (10 eppendorfs of miniprep products).
3. In each tube, put 25 μl of DNA and 5 μl of Master mix.
4. Let digest 2 hours at 37 °C, then incubate 5 minutes at 65 °C.

Aim: Check if the digestion has been done, it would mean that the plasmid contains the insert.

Protocol: follow in this link

What we did in the lab:
Materials:
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
• Colonies B2, E1 and E2
• Electrophoresis cuve
• TAE 1 X
• BET

Method:
1. Make a gel with 1.4 g of agarose, 200 ml of TAE 1 X and 4 droplets of BET.
2. Prepare the samples with 30 µl of digested mix and 6 µl of ladder.
3. Follow the deposit table :
Ladder | Ø | B2 (1) | B2 (2) | B2 (3) | B2 (4) | B2 (5) | B2 (6) | B2 (7) | B2 (8) | B2 (9) | B2 (10) | Ø | E1 (1) | E1 (2) | E1 (3) | E1 (4) | E1 (5) | Ø | Ø | ladder | E1 (6) | E1 (7) | E1 (8) | E1 (9) | E1 (10) | Ø | E2 (1) | E2 (2) | E2 (3) | E2 (4) | E2 (5) | E2 (6) | E2 (7) | E2 (8) | E2 (9) | E2 (10) | Ø | Ø
4. Launch the electrophoresis 20 minutes at 50 V, then 45 minutes at 130 V.

Results:

Aim: Increase the quantity of insert.

Protocol: follow in this link

What we did in the lab:
Materials:
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
• 1.5 ml eppendorfs
• Takara enzyme
• Primers S and AS
• dNTP
• Buffer 6 X
• Distilled water
• MgCl₂

Method:
1. Prepare the following tubes :

Table 2
	Mix with two primers	Mix with primer S only	Mix with primer AS only
Takara enzyme (µl)	6	6	6
Primer S (µl)	6	6	Ø
Primer AS (µl)	6	Ø	6
MgCl₂ (µl)	15	15	15
dNTP (µl)	15	15	15
Buffer (µl)	30	30	30
H₂O (µl)	216	222	222
Total (µl)	294	294	294

2. For each mix, spread 49 µl of it in the samples.
3. Add 1 μl of DNA following the number of tubes :

Table 3
	Mix with two primers	Mix with primer S only	Mix with primer AS only
A1	tube 1	tube 2	tube 3
A2	tube 4	tube 5	tube 6
D1	tube 7	tube 8	tube 9
D2	tube 10	tube 11	tube 12

And tube 13 is the one without DNA
4. Launch the process of PCR.
5. Do an electrophoresis with the results of PCR

Aim: get back the maximum quantity of DNA.
Protocol: follow in this link

What we did in the lab:
Materials:
• Gel of A1⁄A2⁄D1⁄D2
• QIAGEN Extraction gel kit

Method:
Follow the Qiagen gel extraction kit steps with the gel :
A1 : m = 122 mg
A2 : m = 153 mg
D1 : m = 120 mg
D2 : m = 152 mg

Results:

Aim: Get back purified DNA.

Protocol: follow in this link

What we did in the lab:
Materials:
• Gel of B2⁄E1⁄E2
• QIAGEN Extraction gel kit

Method:
Follow the Qiagen Extraction gel kit steps with :

Table 4
DNA	Colonies	Weight of the gel (mg)
B2	Colony 4	367
Colony 7	432
Colony 9	269
E2	Colony 1	300
Colony 2	355
Colony 3	354
Colony 4	314
Colony 5	299
Colony 6	275
Colony 7	277
E1	Colony 1	404
Colony 2	321

Aim: Storage of the inserts.
Protocol: follow in this link

What we did in the lab:
Materials:
• NaAc
• Ethanol 70 %
• Inserts B2⁄E1⁄E2

Method:
1. Use 1/10 volume of NaAc and 2.5 colume of ethanol for each insert :

Table 5
	B2	E1	E2
NaAc (µl)	15	10	35
Ethanol (µl)	875	250	375

2. Resuspend B2⁄E1⁄E2 in 15 µl of H₂O each.
3. We estimated the weight of each inserts :
m(B2) = 240 ng
m(E1) = 60 ng
m(E2) = 420 ng

Aim: Prepare the transformation.
Protocol: follow in this link

What we did in the lab:
Materials:
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
• Inserts B 2 /E 1 /E 2
• pET 43.1 (a+)
• TOP 10 X
• Distilled water
• Ligase

Method:
Use the following volumes :

Table 6
	E1	E2	B2	pET 43.1 (a+)
E1 (µl)	15	Ø	Ø	Ø
E2 (µl)	Ø	15	Ø	Ø
B2 (µl)	Ø	Ø	15	Ø
pET 43.1 (a+) (µl)	4	4	4	4
Ligase (µl)	1	1	1	1
TOP 10 X (µl)	2.2	2.2	2.2	2.2
H₂O (µL)	> Ø	> Ø	> Ø	> 15
> V_total (µL)	> 22.2	> 22.2	> 22.2	> 22.2

Aim: Increase the quantity of DNA.
Protocol: follow in this link

What we did in the lab:
Materials:
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
• Qiagen Miniprep kit
• Digestion enzyme XbaI and HindIII
• Digestion buffer 2.1
• 1.5 ml eppendorfs
• Electrophoresis cuve
• Distilled water

Method:
1. Use the Qiagen kit for our cultures from the 8^th of August :
13 eppendorfs of B1.
20 eppendorfs of C2.
2. Digest the plasmid with the following volumes for each sample :

Table 7
	Volumes (µl)
DNA	5
XbaI	1
HindIII	1
Buffer 2.1	2
H₂O	11
Total	20

3. Analyze the digestion by electrophoresis on an agarose gel prepared with 1.05 g of agarose in 125 ml of TAE 1 X. 4. Launch the electrophoresis, following the deposit table :
C2 | ladder | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | ladder
B1 | ladder | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | Ø | Ø | Ø | 20 | ladder

Aim: Have different clones to know which contain the insert.
Protocol: follow in this link

What we did in the lab:
Materials:
&• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
• Carbenicillin at 50 mg ⁄ ml
• Digestion enzyme XbaI and HindIII
• LB medium
• pET 43.1 (a+)
• C1 v2 colonies
• Shaking incubator (INFORS HT)

Method:
1. Prepare 20 ml of LB with 20 μl of carbenicillin
2. Put 1 ml of this mix in twenty 1.5 ml eppendorfs
3. Take 20 colonies of the petri dish C1 v2 and put them in the previous eppendorfs
4. Let incubate overnight at 37 °C and 150 rpm

Aim: Create a stock of antibiotic.
Protocol: follow in this link

What we did in the lab:
Materials:
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
• Carbenicillin at 50 mg ⁄ ml
• 1.5 ml eppendorfs
• 15 ml falcon
• Distilled water

Method:
1. Prep Put 500 mg of carbenicillin in a 15 ml falcon and 10 ml of distilled water. Then, put the falcon on ice
2. Aliquot the mix in 10 eppendorfs of 1.5 ml
3. Store at −20 °C

Aim: Check if the PCR works.
Protocol: follow in this link

Results:

The PCR works properly since we notive significant bands at the right level.

Aim: After their ligation in TOP 10 cloning, they will be transformed into TOP 10.
Protocol: follow in this link

What we did in the lab :
Materials
• Ligation’s products of A1⁄A2⁄D1⁄D2
&bull ; TOP 10 competent cells
&bull ; SOC
• Microbiology equipement
• Xgal digestion enzyme at 10 mg⁄ml

Method
1. Add 6 μl of ligation product in 50 μl of competent cells.
2. Put the samples 30 minutes on ice, then 40 seconds at 42 °C.
3. Put the samples 3 minutes on ice.
4. Add 150 μl of SOC.
5. Let incubate 40 minutes at 37 °C and 150 rpm
6. Take LB with carbenicillin petri plate and add Xgal to reach 40 μg⁄ml. As the volume is about 25 ml, add 1 mg of Xgal and as the stock solution is 10 mg⁄ml, spread 100 μl on the plate.
7. Spread bacterias on four distincts petri dishes (one for each insert).

Aim: After their ligation we must transform the inserts into bacterias.
Protocol: follow in this link

What we did in the lab :
Method
Add 10 μl of ligation product (to have 100mg) at the beggining.

Aim: Get back the DNA.
Protocol: follow in this link

What we did in the lab :
Method
We use a final volume of 50 μl

Aim: Split the insert and the plasmid.
Protocol: follow in this link

What we did in the lab:
Materials:
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
• Qiagen Miniprep kit
• Digestion enzyme XbaI and HindIII
• Digestion buffer 2 X
• 1.5 ml eppendorfs
• Distilled water
• Shaking incubator (INFORS HT)

Method:
1. Realize a master mix with :

Table 8
	Volumes (µl)
XbaI	20
HindIII	20
Buffer 2 X	40
H₂O	220
Total	300

2. Put μl of the master mix in each of the twenty 1.5 ml eppendorfs and add 5 μl of DNA.
3. Let incubate one hour at 37 °C and 150 rpm, then 5 minutes at 65 °C

Results
The 10^th of August, we realize that we forgot to put XbaI, so we respreads the previous mix on petri dishes with XbaI

Aim: We want to produce 5 μ g of dephosphorylated pET 43.1 (a+) from pET 43.1 (a+) at 400 ng⁄ml and we start with the digestion.
Protocol: follow in this link

What we did in the lab:
Materials:
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
• Qiagen Miniprep kit
• Digestion enzyme XbaI and HindIII
• CutSmart buffer
• 1.5 ml eppendorfs
• Distilled water
• Shaking incubator (INFORS HT)

Method:
1. Put all the following reactants in a 1.5 ml eppendorf and let digest one hour at 37 °C :

Table 9
	Volumes (µl)
DNA	12.5
XbaI	2
HindIII	4
Buffer CutSmart	5
H₂O	26.5
Total	50

2. Inactivate the enzymes 5 minutes at 65 °C.

Aim: Get back the digested and purified plasmid before dephosphorylation.
Protocol: follow in this link

What we did in the lab:
Materials:
• Products from the digestion of pET 43.1 (a+) with HindIII and XbaI
• Agarose
• Electrophoresis cuve
Method:
1. Make a 0.7 % agarose gel
2. Prepare the cuve to do the migration at 100 V with two wells for pET 43.1 (a+) and one for the ladder.
3. Take the results and follow the kit steps of Qiagen extraction kit.
Results>
We notice two bands for digested pET 43.1 (a+), one at 6000bp and another between 1000 and 1500bp. We extract the bands at 6000bp.
We obtained :
m1 = 0.4079 g
m2 = 0 .3720 g

Aim: Check if the digestion works properly and if we have inserts.
Protocol: follow in this link

What we did in the lab:
Materials:
• Products from the digestion of C 1
• Agarose
• Electrophoresis cuve
Method:
1. Take the 20 μl of each sample from the digestion and add 4 μl of loading buffer 6X.
2. Do the electrophoresis, following the deposit table :
Ladder | Ø | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | Ø | ladder
Ladder | Ø | 17 | 18 | 19 | 20
Results
There is no insert. The colonies do not contain our insert, we must redo the experiment with new colonies.

Aim: Transform the bacterias with our recombined plasmid.
Protocol: follow in this link

What we did in the lab:
Method:
The volumes of insert are too small, we add 5 μl of H₂O and diluts at 1⁄100. We realize a transformation in DH3α with 1 μl of DNA and 50 μl of competent cells.

Aim: Have more antibodies.
Protocol: follow in this link

Results
We obtained 30 μl⁄aliquot.

Aim: Increase the quantity of colonies containing inserts.
Protocol: follow in this link

Aim: Ligate the insert and the plasmid.
Protocol: follow in this link

Aim: Transform our inserts in TOP 10 competent cells.
Protocol: follow in this link

Aim: Have bacterias with the right plasmid to produce protein.
Protocol: follow in this link

What we did in the lab:
Materials:
• Digested B1 and C2
• BL21DE3 competent cells
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
• Shaking incubator (INFORS HT)
• SOC
Method:
1. Take 3 samples of C2 (10, 13 and 16) and 2 samples of B1 (6 and 8).
2. Put 1 μl of DNA in 99 μl of H₂O. Then, put 1 μl of DNA (B1 or C2) in 50 μl of BL21DE3 competent cells.
3. Put the samples 30 minutes on ice and then 40 seconds at 42 °C.
4. Put the samples 3 minutes on ice.
5. Add 150 μl of SOC and let incubate 30 minutes at 37 °C and 150 rpm.
6. Spread the mix on a petri dish with LB and carbenicillin.
7. Let incubate overnight at 37 °C and 150 rpm.

Aim: Have the concentration of digested pET 43.1.
Protocol: follow in this link

Results
Measure the concentration of digested pET 43.1 (a+) with a nanodrop. For the first tube, we find a concentration of 6.8 ng⁄μl in 46 μl. For the second tube, we find a concentration of 8.8 ng⁄μl in 46 μl. Then, store the samples at −20 °C.

Aim: Transform C 2 and B 1 in pET 43.1 and DH3 α .
Protocol: follow in this link

Results
For B2 (4, 7, 9), E1 (1, 2) and E2 (2, 3, 4, 5, 6, 7) we see small colonies so we let and 25 μl of carbenicillin.
For B1 and C2 in pET 43.1 (a+), transformations were successful.
For A1, A2 and D1, D2 there is too many bacterias so we take a colony, dilute it in LB and then spread it on petri dish with LB, Xgal and carbenicillin.

Aim: To produce proteins.
Protocol: follow in this link

What we did in the lab:
Materials:
• Spectrophotometer Ultrospec 3100
• iPTG at 0.5 M br/>
Method:
1. Make two measures separated of 30 minutes :

Table 10
Sample	1	2	3	4	5	6	Time of addition of iPTG	Concentration (ng⁄μl)
B1 (1)	0.022	0.062	0.152	0.344	0.557	0.694	16 h 55	0.859
B1 (2)	0.185	0.417	0.693				15 h 25	0.956
B1 (3)	0.075	0.211	0.413	0.688			15 h 55	0.920
C2 (1)	0.060	0.166	0.350	0.627	0.699		16 h 25	0.905
C2 (2)	0.044	0.119	0.296	0.510	0.698		16 h 25	0.910
C2 (16)	0.080	0.230	0.445	0.689			15 h 55	0.907

2. When the DO reach 0.7 keep the last measure and centrifuge 3 minutes at 8000 g.
3. Throw away the supernatent and store at −20 °C.
4. Add iPTG to reach 0.3 mM.

Aim: Make the future ligation easier.
Protocol: follow in this link

What we did in the lab:
Materials:
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
• onSAP
• CutSmart buffer
• 1.5 ml eppendorfs
• Distilled water
• Shaking incubator (INFORS HT)

Method:
Method
1. Start with tube 2 (8.6 ng⁄μl in 46 μl) and use the following mix :

Table 11
	Volumes ( μl)
DNA	46
CutSmart	6
H₂O	6.7
onSAP	1.3
Total	60

2. Let incubate 30 minutes at 37 °C then 5 minutes at 65 °C.
3. Do the same for tube 1.

Aim: Get back the DNA.
Protocol: follow in this link

What we did in the lab

What we did in the lab:
Materials:
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
• LB medium
• Carbenicillin at 50 mg⁄ml
• B1⁄E1⁄E2 in TOPO
Method
We do 31 precultures with a mix with 1 ml of LB and 1 μl of carbenicillin to have :
3 samples of E1
14 samples of B2
14 samples of E2

Aim: Send our insert for sequencing as the transformations in BL21DE3.
Protocol: follow in this link

What we did in the lab

Aim: Have precultures to redo the cultures in 4 x 1 l of LB as they grow well.
Protocol: follow in this link

What we did in the lab

Aim: Increase the quantity of plasmid for the next ligation.
Protocol: follow in this link

What we did in the lab

Aim: Increase the quantity of DNA.
Protocol: follow in this link

What we did in the lab

Aim: Check if the colonies we took contain the insert.
Protocol: follow in this link

What we did in the lab

What we did in the lab
Materials
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
• Digestion enzyme XbaI and HindIII
• Digestion buffer 2.1
• 1.5 ml eppendorfs
• Distilled water
• Shaking incubator (INFORS HT)
• Inserts B2⁄E1⁄E2

Method
1. In a 1.5 ml eppendorf, put :

Table 12
	Volumes (μl)
DNA	5
XbaI	1
H₂O	11
HindIII	1
Buffer 2.1	2
Total	20

2. Let incubate one hour at 37 °C , then 5 minutes at −20 °C.

Aim: Produce the protein in higher quantity.
Protocol: follow in this link

What we did in the lab
Materials
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
• iPTG
• Carbenicillin at 50 mg⁄ml

Method
1. In a 2 L erlenmeyer, put 1 l of LB and 1 ml of carbenicillin and incubate at 37 °C and 150 rpm to warm the liquid.
2. For each preculture of 25 ml, put it in a 50 ml falcon, centrifuge 5 minutes at 3500 rpm, throw away the supernatent and resuspend the pellet in 5 ml of clean LB.
3.Centrifuge 5 minutes at 3500 rpm, throw away the supernatent and resuspend the pellet in 20 ml of clean LB.
4. In each 2 l erlenmeyer, add 5 ml of our preculture : 4 erlenmeyer for C2 and 4 for B1.
5. Let incubate at 37 °C ann 150 rpm and measure the DO.
6. Once the DO reaches 0.7, add 1 ml of iPTG.
7. Let incubate for 3 hours.
8. Centrifuge the cultures.
9. Resuspend the pellet in 10 ml of lyse buffer in a 50 ml falcon of mass known.
10. Store at −20 °C.

Aim: Check if the digestion works.
Protocol: follow in this link

What we did in the lab
Materials
• Agarose
• Qiagen kit
• Nanodrop
• Electrophoresis cuve
• Loading buffer 6X

Method
1. Add 10 μl of loading buffer 6X to reach 50 μl for each sample.
2. Do an electrophoresis and extract the bands of the palsmid digested. We obtaines two tubes :
Tube1 : m1 = 1.276 − 0.9964 = 131.2 mg
Tube2 : m2 = 1.1524 − 0.9994 = 153.0 mg
3. Follow the Qiagen kit steps for a final volum of 50 μl.
4. Measure the concentration with the Nanodrop to see the results.
5. Store at −20 °C.

Results
Tube 1 : 20 .2 ng⁄μl
Tube 2 : 24.8 ng⁄μl

Team:Pasteur Paris/Microbiology week10

click on the weeks

Week 10

August 8, 2016:

150. Culture for miniprep of C2 v2 and B1 v2

151. Digestion of B2, E1 and C2 from the 4th of August

152. Electrophoresis with the results of digestion

153. PCR of inserts A1⁄A2⁄D1⁄D2

154. Gel extraction of A1⁄A2⁄D1⁄D2

155. Gel extraction of B2, E1 and E2

156. Resuspension of inserts B2⁄E1⁄E2

August 9, 2016:

157. Ligation of inserts B2⁄E1⁄E2 with pET 43.1a(+)

158. Miniprep of C2 v2 (TOP 10) and B1 v2 (TOP 10 )

159. Miniprep preculture of C1 v2 in pET 43.1a(+)

160. Preparation of 10 aliquots of carbenicillin at 50 ng⁄m

161. Electrophoresis of the PCR done on the 8th of August with A1⁄A2⁄D1⁄D2

162. Transformation of A1⁄A2⁄D1⁄D2 in TOP 10

164. Transformation of E1⁄E2 and B2 in TOP 10

August 10, 2016:

165. Miniprep of C1 v2 (culture from the 9 of August)

166. Digestion of C1 v2 before electrophoresis

167. Digestion of pET 43.1 (a+) with XbaI and HindIII

168. Electrophoresis and gel extraction

169. Electrophoresis of C1 digested

170. Transformation of B2 (4, 7 and 9), E1 (1 and 2) and E2 (2, 3, 4, 5, 6 and 7)

171. Aliquot of antibodies 462

172. Culture of A1⁄A2⁄D1⁄D2

173. Ligation of A1⁄A2⁄D1⁄D2 in TOPO

174. Transformation of A1⁄A2⁄D1⁄D2 with TOPO in TOP 10 competent cells

175. Transformation of B1 and C2 in BL21DE3

176. Dosage of digested pET 43.1 (a+)

177. Transformation of C2 and B1 in pET 43.1a(+) and DH3α

August 11, 2016:

178. Absorbance of precultures C2 (1, 2, 3) and B1 (1, 2, 16)

179. Dephosphorylation of pET 43.1a(+) digested on the 10th of August

180. Miniprep of B1⁄E1⁄E2 in TOPO

181. Transformation of B1 colony 8 and C2 colony 16 in pET 43.1 (a+) and in DH 3α

182. Precultures of B1 v2 and C2 v2

183. Digestion of pET 43.1 (a+) with XbaI and HindIII

August 12, 2016:

184. Miniprep from precultures of B2⁄E1⁄E2 in TOPO

185. Digestion of inserts B2⁄E1⁄E2 with XbaI and HindIII

186. Culture of C2 and B1 in 1 l of LB

187. Agarose gel to analyse digestion of pET 43.1 (a+) done on the 11th of August

151. Digestion of B2, E1 and C2 from the 4^th of August

161. Electrophoresis of the PCR done on the 8^th of August with A1⁄A2⁄D1⁄D2

179. Dephosphorylation of pET 43.1a(+) digested on the 10^th of August

187. Agarose gel to analyse digestion of pET 43.1 (a+) done on the 11^th of August