Difference between revisions of "Team:ECUST/Experiment"

Materials:

5xl Gxl Buffer
Primer star Gxl（DNA polymerase）
dNTP
ddH₂O
Rhodobacter sphaeroides 2.4.1 genome
plasmid backbone with sYFP2 gene

Forward/Reverse Primer：

Method:

PCR program:

PCR System（50μL）：

Materials:

PCR fragment (concentration requirement> 100 ng / μL)
Linearized plasmids (concentration requirements> 100 ng / μL, can be prepared by digestion or reverse PCR)
1.33x ITA reagent

Method:

1. Add all kinds of PCR fragments and linear plasmids of the same mass to 1.33x ITA reagents (7.5 µL), making total volume 10 µL
2. Keep the system at 50°C for 1h
3. Transform 10 µL ligation liquid and select using chl resistance

Materials:

10xl Taq Buffer
Taq polymerase
dNTP
ddH₂O

Forward/Reverse Primer：

Method:

1. Select monoclonal strains to LB plate with chl resistance, and carry out PCR after cμLture for 2h
2. PCR

PCR system（10μL）：

5xl Taq Buffer	2 µL
dNTP	0.8μL
Taq polymerase	0.2 µL
Forward primer	0.5 µL
Reverse primer	0.5 µL
template	1 µL
ddH2O	5 µL

PCR procedure


3. Examine the resμLts using electrophoresis. If positive, inocμLate 5 mL to LB plat with chl resistance for overnight cμLture. Preserve the stains with 15% glycerol.

Materials:

Rhodobacter sphaeroides 2.4.1
Ecoli-sm10
Joint film
LB medium
PBS buffer

Method:

1. Ecoli-sm10, Rhodobacter sphaeroides 2.4.1 were incubated overnight for 12 h. Add the corresponding antibiotics to donor bacteria .
2. Ecoli-sm10, Rhodobacter sphaeroides 2.4.1 secondary cμLture, add the appropriate antibiotic into the donor bacteria medium, shake to the logarithmic growth phase.
3. Take Ecoli-sm10, Rhodobacter sphaeroides 2.4.1, each 1 mL wash with PBS and mix them, take 25 μL drop in the dried adhesive film, dry. CμLtivate overnight.
4. Wash the bacteria moss on the joint membrane with 1 mL of medium or PBS and take 100 μL of the coated plate. The resμLts were observed after several hours of cμLture.
5. After one screening, it was induced with TSB containing 10% sucrose and then subjected to secondary screening on a plate containing 10% sucrose free of resistance.
6. Validate gene knockout and gene knocking by priming pairs on the genome.

Materials:

5xl Gxl Buffer
Primer star Gxl（DNA polymerase）
dNTP
ddH₂O
pIND4 plasmid
pMCS-eq plasmid

Forward/Reverse Primer：

Method:

Mentioned above.

Mentioned above.

Materials:

Enzyme NcoI and BamHI
10xBuffer（NEB buffer 1.1 2.1 3.1 star）
PCR DNA
ddH₂O
pIND4 plasmid DNA

Method:

1. Configuration of double enzyme digestion system：

Digestion System（50μL）：

NcoI	1 μL
BamHI	1 μL
10 x Buffer	5 μL (NEB's buffer score 1.1 2.1 3.1 star specific see enzyme instructions select the cutting efficiency and asterisk the best activity)
ddH2O	added as appropriate
DNA	1 μg

2. the reaction temperature of 37 ℃ 15min (specific reaction time see the instructions).
3. After the end of the reaction 80 ℃ 20min inactivation.

Materials:

T4 DNA ligase
10x buffer
Plasmid DNA
PCR DNA
ddH₂O

Method:

1. According to the external source and carrier concentration ratio of 5: 1 configuration connection system 20μL

Ligation System（20μL）：

10 x buffer	2 μL
T4 DNA ligase	1 μL
plasmid DNA	X μL
PCR DNA	Y μL

2. 16 ℃for 1 h
3. The connection solution to take 10μL to transform, with kanr resistance screening positive bacteria

Materials:

10% glycerol
2mm Electroporation cup
Kanr plate
ddH₂O

Method:

1. 32℃ overnight cμLture and transfer, until the OD grows to about 2.5
2. 10 mL of bacteria at 5000 rpm for 5 min to collect all the cells
3. 2 mL sterile water pumped retrogradesum (washed away from the medium ion residue, centrifuged at 5000 rpm for 2 min)
4. with 200μL 10% glycerol resuspend the bacteria
5. 100μL of competent and 100ng plasmid mixed
6. by adding 2mm electric rotor 1.5Kv shock (voltage index curve mode, capacitance 25uF, resistance 200Ω)
7. 600 μLTSB mixed and moved into the EP tube
8. 32℃, 220rpm recovery 2h
9. 5000 rpm after centrifugation, remove the 400μL supernatant, the remaining bacteria coated on kanr resistant plate
10. cμLture 2-3d observation resμLts

Materials:

Media for fermentation ：
For 1L media:
Succinic acid disodium salt 5.49g
NaCl 0.4g
MgS04·7H20 0.2g
CaCL2·2H20 0.05g
L-glutamate 5mol
KH2PO4 1.0g
K2HPO4 ·2H20 1.572g
Yeast extract fermentation 1.0g
Solution of trace element 1.0 mL
Solution of growth factor 1.0 mL
For 100 mL solution of trace element:
0.21 g MnSO4 · 4H2O
0.28 g H3BO3
0.004 g Cu(NO3)2 · 7H2O,
0.024 g ZnSO4 · 7H2O,
0.075 g Na2MoO4 · 2H2O


For 100 mL solution of growth factor:
0.01g biotin
0.5g thiamine HCl
1.0g nicotinic acid

Main protocol:

1. Rhodobacter sphaeroides 2.4.1 were cμLtured in TSB medium for 48h
2. Took 10 mL bacterial suspension and removed the clear supernatant extract after demission at 3500rpm for 5 minutes
3. Resuspended the bacteria in 10 mL fermentation medium and transferred them into a new conical flask
4. Added 90 mL fermentation medium into the flask with 100μL solution of growth factor, 100μL solution of trace element, 30ng/μL kanamycin and 100μL IPTG (making the final concentration 800μL)
5. Introduced argon to the flask through rubber tube for 5 min and wrapped the bottleneck with plastic membrane in case of gaseous escape
6. Pressed the bottleneck with rubber stopper and sealed it with parafilm
7. CμLtured the bacteria in the flask which was exposed to 4,000lx under anaerobic condition with nutrient at 32℃
8. Collected the gas based on water gas displacing principle using pinhead through rubber stopper and the pinhead shoμLd be covered with Vaseline
9. Drew bacterial suspension with syringe and measured OD every 24 hours. After each measurement, the pinhead shoμLd be covered with Vaseline.