Team:Pasteur Paris/Microbiology week5

Aim:We do again the previous experiment to understand why transformation in our bacteria does not work.

Protocol: follow in this link

What we did in the lab:
Materials:
• pET43.1a(+)a plamid (obtained with Midiprep done on June 8, 2016)
• enzyme restriction (XbaI / HindIII)
• Buffer Cutsmart 10X (NEB)
• H2O
• P10 pipet, P20 pipet, 1.5 mL eppendorf, 37°C and 65°C water bath

Method:
1. For our manipulation, we used XbaI and HindIII enzymes and we used Cutsmart 10 X buffer because it is the most appropriate buffer.
2. Follow the next table to volumes:

	pET43.1a(+) à 130 ng/µl (3 µg)
DNA (µl)	19
XbaI (µl)	2
Hind III (µl)	1
H20 (µl)	5
CutSmart 10X (µl)	24.5
TOTAL (µl)	51.5

Table 35

	= 260 nm	pET43.1a(+) XbaI/HindIII
N°1	A260/280	1.8
N°1	Cfinal	260 ng/µL
N°2	A260/A280	1.4
N°2	Cfinal	400 ng/µL

Table 36

Protocol: follow in this

Aim:Check if digestion was successfully done

Protocol: follow in this link

What we did in the lab:
Materials:
• pET43.1a(+) plasmid (obtained with Midiprep on june 8, 2016)
• pET43.1a(+) plasmid digested by XbaI/HindIII
• gel 0.7% agarose
• TAE 0.5X buffer
• Electrophoresis generator ( at 50 V and after at 90 V)
• DNA ladder (Thermoscientific gene ruler 1kb)
• P10 and P20 pipet, 1.5 eppendorf,
electrophoresis BIORAD Mini-Sub Cell GT
Method:
1- Prepare an agarose gel at 0.7 % (Refer on the detail protocol which is on the protocol parts)
2- Fill electrophoresis chamber with TAE 1X
3- Follow the deposit Table 37:

Line	L1	L3	L5
Name	Marker weight	pET43.1a(+) X/H	pET43.1a(+) UNCUT
A_{DNA (µl)}	5	10	5
H20 (µl)	0	0	0
Load buffer 6X	0	2	1

Table 37

1- Put the voltmeter at 100 V for one hour
Results:

IMAGE Figure 7

We saw that in the line of the plasmid cut by XbaI and HindIII, we have two bands which don’t correspond to the uncut plasmid band. Moreover, The band of the plasmid cut by XbaI and HindIII have the appropriate weight. So we can cuclude that the digestion has worked. We can get back our plasmid cut with extraction gel.

Aim:To recover the pET43.1a(+) which has been digested, and to purify out the band from the gel.

Protocol: follow in this link

What we did in the lab:
Materials:
• Results of electrophoresis (done previously)
• Gel extraction kit from Qiagen
• P10 and P20 pipet, 1.5 ml Eppendorf
Method:
To extract DNA we use the Gel Extraction Kit from Qiagen and we follow the different steps detailed in the kit.

• Eppendorf mass : 1.4043 g
• Eppendorf + Gel mass : 1.0817 g
• Gel mass : 1.4043 – 1.0817 = 322.6 mg

We must pour 3 volums of QG buffer, it means 967.8 µl.
We added 322 µl of isopropanol and we split in two equals volums our experiment

	DNA Masses (mg)	DNA Volumes (ml))
pET43.1a(+)	322.6	1.073

Table 38

Aim:After the electrophoresis, we saw that the digestion of pET43.1a(+) was done succesfully. Now we have to dephosphorylate it to avoid self-ligation.

Protocol: follow in this link

What we did in the lab:
Materials:
• pET43.1a(+) plasmid digested by XbaI/HindIII
• Dephosphorylation rSAP enzyme
• P10 and P200 pipet, 1.5 eppendorf, water bath (37 °C and 65 °C)
Method:
1. For volumes, refer on the next table :
We dephosphorylated 7.5*120 ng/µl, so 900 ng of pET43.1a(+)

DNA of pET43.1a(+)( µl)	rSAP ( µl)	Buffer 10X ( µl)	H2O ( µl)	TOTA ( µl)
7.5	2.58	6	43.92	60

Table 39

Aim:To save time, we do another dephosphorylation of pET43.1 digested by enzymes to ligate it to the insert C2 (digest by XbaI/HindIII on June,28 2016).

Protocol: follow in this link

What we did in the lab:
Materials:
• pET43.1a plasmid digested by XbaI/HindIII
• Dephosphorylation rSAP enzyme
• P10 and P200 pipet, 1.5 eppendorf, water bath (37 °C and 65 °C)
Method:
1. For volumes, refer on the next table: 2. We dephosphorylate 7.5*120 ng/µl, so 900 ng of pET43.1

DNA of pET43.1a(+)( 120 ng/µl)	rSAP ( µl)	Buffer 10X ( µl)	H2O ( µl)	TOTA ( µl)
7.5	2.58	6	43.92	60

Table 39

Aim:We want to obtain a second expression vector, this time with pET43.1 and C2. We do the same experiment as previously performed for C1.

Protocol: follow in this link

What we did in the lab:
Materials:
• pET43.1a(+) plasmid digested by XbaI/HindIII and dephosphorylated
• C2 insert cut by XbaI/HindIII (done on the June,28 2016)
• T4 Ligase and Buffer 10X
• P10 and P200 pipet, 1.5 eppendorf, waterbath (37 °C and 65 °C)
Method:
1. For volumes, refer to the next table:
C2 = 9.2 ng/µl
We used 60 µl of pET43.1a(+) concentrated at 15 ng/µl (total 900 ng)

	1:1	1:3	Only pET43.1a(+)
pET43.1a(+) (50 ng)	6.7	6.7	6.7
Insert (C2) (µl)	1.74	5.22
T4 Ligase (µl)	1	1	1
Buffer 10X (µl)	1.5	1.5	1.5
H20 (µl)	4.06	0.58	5.8
TOTAL (µl)	15	15	15

Table 40

2. Incubate 10 min at room temperature, and put it at 65 °C during 10 min to inactivate the ligase. To keep it and use it after, we put it at -20 °C

Results:

Aim:At this stage, we want to obtain a recombinant vector (pET43.1a(+) + C2)

Protocol: follow in this link

What we did in the lab:
Materials:
• pET43.1a(+) plasmid digested by XbaI/HindIII and dephosphorylate (done previously)
• C2 insert cut by XbaI/HindIII (done on the june,28 2016)
• T4 Ligase and Buffer 10X
• P10 and P200 pipet, 1.5 eppendorf, warm bath (37 °C and 65 °C)
Method:
1. For volums, refer to the next table :

C2 = 11.4 ng/µl
pET43.1 = 50 ng/µl (900 ng 60 µl)>

	1:1	1:4	Only pET43.1a(+)
pET43.1a(+) (50mg) (µl)	20	20	20
Insert C2 (µl)	1.4	4.2
T4 ligase (µl)	1	1	1
Buffer 10X (µl)	2.5	2.5	2.5
H20 (µl)	0	0	0
TOTAL (µl)	24.9	27.7	22.5

Table 42

Incubate 10 min at room temperature, and put it at 37 °C during 10 min. To keep it and use it after, we put it at -20 °C.

Aim:Check if ligation of pET43.1+C1 and pET43.1+C2 were successfully done.

Protocol: follow in this link

What we did in the lab:
Materials:
• pET43.1a plamid
• pET43.1a plamid cutted by HindIII/XbaI
• C1 and C2 cutted by HindIII/XbaI
• agarose gel 0.7%
• TAE 0.5x buffer
• Electrophoresis generator at 130 V
• DNA ladder (Thermoscientific gene ruler 1 kb)
• Electrophoresis generator (at 50 V and after at 90 V)
Method:
1. Prepare an agarose gel at 0.7 % (Refer on the detail protocol which is on the protocol parts)
2. Fill electrophoresis chamber with TAE 0.5X
3. Follow the deposit table :

Lanes	L1	L2	L4	L5	L7	L8	L9
Name	Marker weight	pET43.1a(+) uncul	C1 (1:1)	C1 (1:3)	C2 (1:1)	C2 (1:3)	pET43.1a(+) only
DNA (µl)	7	6	5	5	5	5	5
DNA (µl)	0	0	0	0	0	0	0
DNA (µl)	0	0	1	1	1	1	1

Table 43

Let 1h at 50V and put it at 100 V.

Results:

The electrophoresis shows us that DNA wasn’t ligated, so we added 1 µl more of T4 ligase and 1 µl of buffer in each tube and we let ligate during 1h.

After one hour, we did another electrophoresis.

Aim:Check if ligation of pET43.1+C1 and pET43.1+C2 was successfully done.

Protocol: follow in this link

What we did in the lab:
Materials:
• pET43.1a plamid
• pET43.1a plamid cutted by HindIII/XbaI
• C1 and C2 cut by HindIII/XbaI
• agarose gel 0.7%
• TAE 0.5x buffer
• Electrophoresis generator at 130 V
• DNA ladder (Thermoscientific gene ruler 1 kb)
• P10 pipet, P20 pipet, test tube 250mL, electrophoresis BIORAD Mini-Sub Cell GT, 2 type of tips, 1.5ml Eppendorf sterile tubes, 37°C water bath, shaking incubator centrifuge 5415D,

Method:
1. -Fill the electrophoresis chamber with TAE 0.5X buffer
2. Following this deposit table :

Lanes	L1	L3	L4	L5	L6	L7	L8	L9
Name	Marker weight	pET43.1a(+) uncul	pET43.1a(+) only	C1 (1:1)	C1 (1:3)	C2 (1:1)	C2 (1:3)
DNA (µl)	5	1	5	5	5	5	5	5
DNA (µl)	0	0	0	0	0	0	0	0
DNA (µl)	0	1	1	1	1	1	1	1

Table 44 Results:

Results:
MpET43.1 = 5 300 bp * 660 g.mol-1.bp-1
MpET43.1 = 3.5*10-6 g/mol
After condensation, we lost 5 299 molecules of water (one between each bp), so we have 0.1*106 g/mol

Finally, we have :
MpET43.1 = 3.4*106 g/mol
Minsert = 5.6*105 g/mol

According to the instruction:
- We have 0.12 µmol/L in 20 µl, and we must have 10 pg to have a good yield in colonies.
- Finally, we have 0.12 * 20*10-6 = 2.4*10-6 µmol of plasmid and 8.16 pg of plasmid.

Aim:: To make stock cells containing the plasmids, we need to enter the plasmid with C1 and C2 insert into the bacteria DH5 competent cells.

Protocol: follow in this link

What we did in the lab:
Materials:
competent cells, SOC media, 42°C waterbath, LB/carbenicillin 50 µg/ml.
Method:
1- In five 1.5 ml eppendorf tubes, we put 40 µl of DH5 competent cells and we add 5µl of :
(vector:Insert) ratio
(1) (1:0) empty plasmid
(2) C1 (1:1)
(3) C1 (1:3)
(4) C2 (1:1)
(5) C2 (1:3)

2- Place the tubes on ice during 30 min (18h30)
3- Put them at 42 °C during 40 sec
1- Put them on ice during 2 min 30 sec
2- Add 200 µl of SOC in each tubes and place them in a shaking incubator for 1 h at 37 °C and at 225 RPM
3- We spread our mix (250 µl) on petri dishes made of LB and carbenicillin

4- Put them at 37 °C for one night
Results:

Only two colonies have grown on the plate C2 (1:3), all the other petri dishes are empty.
According to the electrophoresis gel, the ligation has worked for C1 (1:3) and C2 (1:1) but it do not correspond to our results. We decided to pool C2 (1:1) with C2 (1:3) and C1 (1:1) with C1 (1:3) because the gel shows the same efficiency of ligation, in order to have more DNA for transformation.

Aim:We want to increase the number of bacteria to check if they have the right plasmid. As we have two colonies, therefore we placed them to grow in liquid media in two Falcons.

Protocol: follow in this link

What we did in the lab:
Method:
In a 50 ml Falcon :
- Put 10 ml of LB and 5 µl of carbenicillin at 100 mg/ml.
- Add the colony we took with a tooth-pick.
- Put the Falcon in a shaking incubator at 37 °C for one day.

Results:

The ligation does not work well, so we pooled our ligation products to increase the amount of available DNA. Aim:: At this stage, we want to obtain a ligated vector (pET43.1a(+) + C1/C2)

Protocol: follow in this link

What we did in the lab:
Method:
C(pET43.1) = 29.5 ng/µl
C(C1) = 11.4 ng/µl
C(C2) = 9.2 ng/µl

Add volumes of 1µl of T4 Ligase and 1 µl of Buffer 10X in the tubes C1(1 :1)+C1(1 :3) and C2(1 :1)+C2(1 :3).
Let the ligation proceed during 1h30 at RT and incubate it 10 min at 65 °C to inactivated the ligase.

Aim:The previuos experiment being still underway, we want to move ahead wier inserts. We want to digest our B2 insert to put it in the expression vector.

Protocol: follow in this link

What we did in the lab:
Materials:
• eppendorf (0.5 ml)
• microbiology equipement
• B2 insert
• Enzymes (HindIII and XbaI)
• H2O RNAse free
• Buffer 10X

Method:
1. Mixing all of reactants and let the digestion proceed during 1h30 at 37 °C
2. Follow this next table

	B2
Vol DNA (50ng/µl)	20 µl
Vol Hind III	0.5 µl
A_{Vol XbaI}	0.5 µl
Vol buffer 10X (CutSmart)	2.5 µl
Vol TOTAL	25 µl

Table 45

Allow the digestion proceed for 1h and add an extra 0.5 µl of enzyme and leave the reaction going for 1h more at 37 °C.
1. Incubate it 10 min at 65 °C to inactivate the enzymes

Aim:Than we do the ligation of the insert B2 with pET43.1 and the transformation of all our products of ligation B2/C1 mix and empty plasmid as control.

(Refer to previous protocol)

Results:

Results:

B2 (1 : 1) Nothing has grown
B2 (1 : 3) Nothing has grown
C2 (1 : 0) Nothing has grown
C1 mix Nothing has grown

Aim:To get the plasmid back and verify that it contains inserts. We have two colonies, so for each colony, we started two cultures in Falcon tubes. One being a primary culture and the other a backup, in case the culture doesn't grow.

Protocol: follow in this link

What we did in the lab:
Materials:
They are named :
C2 (1 :3) 1.1
C2 (1 :3) 1.2
C2 (1 :3) 2.1
C2 (1 :3) 2.2

NB :(The naming convention here is first number is the number of colony, and the second number correspond to primary 1 ou secondary 2 cultures).
Method:
Results:

Protocol: follow in this link

Aim:We want to check if our bacteria have produced enought ligated plasmid.

Protocol: follow in this link

What we did in the lab:
Materials:
Method:
Spectro : Ultrospec 3100 pro-Amersham Bioscience
Vol DNA = 2 l
Vol TE buffer= 498 µl
Dilution = 1/250
In a quartz cuvette ( Path Length= 1 cm):
- 1 ml of buffer TE
- use 2 µl DNA in 998 µl of TE for the dilution
Analysis to = 260 nm
Blank on TE1X
Results:

λ= 260 nm	Colony (1:1)	Colony (1:2)	Colony (2:1)	Colony (2:2)
Cfinale>	110 ng/µl	290 ng/µl	110 ng/µl	70 ng/µl
Cfinale2>	5 500 ng/µl	14 500 ng/µl	5 500 ng/µl	3 500 ng/µl

Table 46

Aim:: We want to check if the plasmid has an insert.

Protocol: follow in this link

What we did in the lab:
Materials:
Method:

	Colony (1:1)	Colony (1:2)	Colony (2:1)	Colony (2:2)
DNA (1 µg)	9.1 µl	3.4 µl	9.1 µl	14.3µl
XbaI	1 µl	1 µl	1 µl	1 µl
HindIII	1 µl	1 µl	1 µl	1 µl
H20	15.9 µl	21.6 µl	15.9 µl	10.7 µl
Buffer 10X	3 µl	3 µl	3 µl	3 µl
Buffer 10X	30 µl	30 µl	30 µl	30 µl

Table 47

1. Add all reagents in a 1.5 ml eppendorf
2. Let the digestion proceed during 1h30 at 37 °C and incubate 10 min at 65 °C
3. For the reagent volumes, refer to the table

Aim:We want to check if the plasmid has an insert.

Protocol: follow in this link

What we did in the lab:
Method:

	Lanes	L1	L2	L3	L4	L5	L6	L7	L8	L9	L10
Name	Mark weight		pET43.1a(+) X/H		C2 (1:1)		C2 (1:2)		C2 (2:1)		C2 (2:2)	pET43.1a(+) uncut
DNA (µl)	6		30		30		30		30		30	30

Table 48

FIGURE

Results:

After the elctrophoresis, we notice that the recombinating plasmid seems to contain two inserts. Indeed, the band corresponds to a size between 1 00 and 1 500 bp.
We decided to digest our double insert with XbaI and SpeI to split it.
We also digested a pET43.1with XbaI and SpeI to fit it.

Aim:We observed that two inserts were present in our gels, we therefore want to verify that the second heavier one is not as a result illegitimate ligation of XbaI/HindIII. There is a SpeI site in the neighboring sequences. Therefore, we want to split the insert to have just one insert.

Protocol: follow in this link

What we did in the lab:
Materials:
Method:
1. For volumes, refer to the next table :

	C2.1.1 (110 ng/µl)	C2.1.2 (290 ng/µl)	pET43.1a(+)
Vol DNA (3 µl)	27.3 µl	10.3 µl	2.5 µl
Vol SepI (µl)	1	1	1
Vol XbaI (µl)	1	1	1
H20 10X (µl)	6.7	23.7	31.5
Vol Buffer CutSmart 10X (µl)	4	4	4
Vol TOTAL (µl)	40	40	40

Table 49

2. After 1H of digestion, we added 1 µl of each enzyme and let digest 30 more minutes.

Aim:We want to check if we succeeded in splitting the twinned insert.

Protocol: follow in this link

What we did in the lab:
Method:
1. To deposit volums, refer to this table :

Lines	L1	L3	L5	L7
Name	Mark weight	pET43.1 X/S	C2 (1:1)	C2 (1:2)
DNA (µl)	10	40	40	40
H20 (µl)	0	0	0	40
H20 (µl)	10	10	10	10

Table 50

Results:The digestion does not work, we did not succeed in splitting the twinned insert so we decided to keep it and to express the protein with the double insert, hoping that the stop codon at the end of one of the inserts will be ennough. We will also sequence the plasmid to verify the orientation, and validity of our twinned insert hypothesis.

Aim:In this part we wantant to proceed with the expression of our fusion protein containing the R5-CBD-BPA. To do this we need a cell line having a T7 RNA polymerase. The cells we chose were BL21De3 pLys S. These contain the T7 phage producing the T7 RNA polymerase, and lysozyme, which will inhibit the T7 RNA polymrase and help control the expression better in case our protein is toxic to the cells, and secondly in case the cells escape our control they will not be able to survive in nature, as they will lyse over time.

Protocol: follow in this link

What we did in the lab:
Materials:
BL21De3 competent cells, SOC media, 42 °C waterbath.
Method:
(I) : 50 µl of bacteria + 5 µl of pET43.1-C2 1.1
(II) : 50 µl of bacteria + 5 µl of pET43.1-C2 1.2
(III) : 50 µl of bacteria + 5 µl of pUC
(IV) : 50 µl of bacteria + 5 µl of CT (plasmid given with the bacteria)

After heat shock at 42°C, the cells were allowed to recover by growing at 37°C for 40 minutes in 150 µl of SOC, then the 200 µl of each sample were spread on a petridish with carbenicillin and grown at 37 °C for one night.

Team:Pasteur Paris/Microbiology week5

click on the weeks

Microbiology Notebook

Week 5

July 4, 2016:

47.Digestion of pET43.1 (a) with XbaI and HindIII

48. DNA measurements

49. Agarose gel (0.7 % agarose gel )

50. Electrophoresis on agarose gel of pET43.1a(+) digest by XbaI and HindIII

51. Extraction gel of pET43.1a(+) digest by XbaI and HindIII

52. Dephosphorylation of pET43.1a(+) (digest by XbaI/HindIII) done previously

July 5, 2016:

53. Dephosphorylation of pET43.1a(+)digest by XbaI/HindIII)

54. Ligation of dephosphorylated pET43.1a(+) with C2

55. Ligation of pET43.1a(+) dephosphorylate with C2

56. Electrophoresis on agarose gel of pET43.1 digest by XbaI and HindIII

57. Electrophoresis of pET43.1a(+) digested by XbaI and Hind III

58. Plasmid concentrations

59. Transformation of DH5 competent cells

July 6, 2016:

60. Verification of transformation of the 5/07/16

61. Cultivating colonies to recover the ligated plasmids-C1, or C2 inserts

62. Ligation of pET43.1a(+) with C1 and C2

63. Digestion of insert B2

64. ligation of insert B2 in pET43.1a(+)

July 7, 2016:

65. Check of the petri dish done on the July 6, 2016

66. Extraction of DNA of colonies from 06/07/2016

67. Agarose gel

68. Dosage of extracted DNA

69. Digestion of extracted DNA

70. Electrophoresis of pET43.1a(+)

71. Digestion of the recombinating plasmid

72. Electrophoresis of our results

July 8, 2016:

73. Transformation of bacteria BL21DE3