• pET43.1a(+)a plamid (obtained with Midiprep done on June 8, 2016)
• enzyme restriction (XbaI / HindIII)
• Buffer Cutsmart 10X (NEB)
• H2O
• P10 pipet and 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(+) 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:
Table 40
Line
L1
L2
L3
L4
L5
Name
Marker weight
pET43.1a(+) X/H
pET43.1a(+) UNCUT
ADNA (µl)
5
10
5
H20 (µl)
0
0
0
Load buffer 6X
0
2
1
1- Put the voltmeter at 100 V for one hour
Results:
Figure 7 : Digestion gel of pET43.1(a+)
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.
• 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
Table 41
DNA Masses (mg)
DNA Volumes (ml))
pET43.1a(+)
322.6
1.073
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.
• 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)
Table 43
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
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
Aim:
At this stage, we want to obtain a recombinant vector (pET43.1a(+) + C2)
• 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 in 60 µl)
Table 44
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
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.
• 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 :
Table 45
Lanes
L1
L2
L3
L4
L5
L6
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
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 1 hour.
After one hour, we did another electrophoresis.
Aim:
Check if ligation of pET43.1+C1 and pET43.1+C2 was successfully done.
• 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 :
Table 46
Lanes
L1
L2
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
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.
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 (6:30PM).
3- Put them at 42 °C during 40 sec.
4- Put them on ice during 2 min 30 sec.
5- 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.
6- We spread our mix (250 µl) on petri dishes made of LB and carbenicillin.
7- Put them at 37 °C for one night.
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.
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.
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)
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 previous 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.
• eppendorf (0.5 ml)
• • Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
• 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
Table 47
B2
Vol DNA (50ng/µl)
20 µl
Vol Hind III
0.5 µl
AVol XbaI
0.5 µl
Vol buffer 10X (CutSmart)
2.5 µl
Vol TOTAL
25 µl
1. 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.
2. 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.
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.
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.
Aim:
We want to check if our bacteria have produced enought ligated plasmid.
What we did in the lab:
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:
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.
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.
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.
• 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.
