Team:SUSTech Shenzhen/Notebook/Biobricks

Team SUSTC-Shenzhen

Biobricks

Notebook

Contents

8 Chromoprotein

Plasmid Construction

ALL ABBREVIATIONS USED:

Name Number Index Position Backbone Length
Strong promoter
BBa_J23100 4-17-D BBa_J61002 35
Weak promoter
BBa_J23106 4-17-P BBa_J61002 35
Strong RBS ③ BBa_B0034 4-1-N pSB1A2 12
Weak RBS ④ BBa_B0031 2-2-H pSB1C3 14
Terminator ⑤ BBa_B0015 3-3-F pSB1C3 129
eforRed ⑥ BBa_K592012 6-15-I pSB1C3 681
gfasPurple ⑦ BBa_K1033919 6-9-K pSB1C3 669
FwYellow ⑧ BBa_K1033910 4-6-K pSB1C3 714
SpisPink ⑨ BBa_K1033932 6-11-k pSB1C3 678
①+③ ⑩ BBa_K880005 2-3-F pSB1C3 55
Medium promoter+④ 11 BBa_K608007 1-5-G pSB1C3 57

7.22

Transformation of ①, ③, ⑤

Time: 20:30

Handler: Liao Weiduo

Procedure: IGEM Protocols

1. Thaw competent cells on ice.

2. Punch a hole into the corresponding hole in the hole in 2016 kit plate by a 10 ul pipette tip. Adding 10ul ddH­2O to the hole and pipette up and down for several times. Let sit for several minutes to make sure the dyed DNA is fully resuspended.

3. Pipette 50ul of competent cells into 1.5ml tube.

4. Pipette 1ul of resuspended DNA into 1.5ml tube.

5. Pipette 1ul of ddH2O as a control into 1.5ml tube.

6. Close 1.5ml tubes, incubate on ice for 30 min.

7. Heat shock tubes at 42℃ for 1 min.

8. Incubate on ice for 5 min.

9. Pipette 200ul SOC media to each transformation.

10. Incubate at 37℃ for 2 hours, 220rpm.

11. Pipette each transformation on petri plates for a 100ul, and spread with sterilized spreader immediately.

12. Incubate transformations overnight at 37℃.

7.23

Picking single colonies of ①, ③, ⑤

Time: 16:00

Handler: Liao Weiduo

Procedure:

  1. Pick single colonies, adding 6ml LB with corresponding antibiotics, each plate we pick 2 single colonies, A and B.
  2. Incubate overnight at 37℃, 220rpm.

7.24

Plasmid extraction of ①, ③, ⑤ A and B

Time: 9:00

Handler: Liao Weiduo

Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol

  1. Add 2ml LB medium to a 2ml microcentrifuge tube.
  2. Centrifuge at 10,000 g for 1 minute at roonm temperature.
  3. Discard the supernatant.
  4. Repeat Step1-3 until 4ml sample has been transferred to the 2ml tube.
  5. Add 250ul Solution I/RNase A. Vortex to mix thoroughly.
  6. Add 250ul Solution II. Invert and gently rotate the tube several times to obtain a clear lysate. A 2-3 minute incubation may be necessary.
  7. Add 350ul Solution III. Immediately invert several times until a flocculent white precipitate forms.
  8. Centrifuge at maximum speed (>13,000 g) for 10 minutes.
  9. Insert a HiBind DNA Mini Column into a 2ml Collection Tube.
  10. Transfer the cleared supernatant (~830ul)from Step 8 by CAREFULLY aspirating it into the HiBind DNA Mini Column.
  11. Centrifuge at maximum speed for 1 minute.
  12. Discard the filtrate and reuse the collection tube.
  13. Add 500ul HBC Buffer. (HBC Buffer must be diluted with isopropanol before use)
  14. Centrifuge at maximum speed for 1 min.
  15. Discard the filtrate and reuse collection tube.
  16. Add 700ul DNA Wash Buffer. (DNA Wash Buffer must be diluted with 100% ethanol prior to use.)
  17. Centrifuge at maximum speed for 1 min.
  18. Discard the filtrate and reuse the collection tube.
  19. Repeat Steps 16-18.
  20. Centrifuge the empty HiBind DNA Mini Column for 2 minutes at maximum speed to dry the column matrix.
  21. Transfer the HiBind DNA Mini Column to a clean 1.5ml microcentrifuge tube.
  22. Add 80ul ddH2O.
  23. Let sit at room temperature for 2 min.
  24. Centrifuge at maximum speed for 1 min.

Results:

‘’’Concentration:’’’ ①A: 245.9 ng/ul 1.84; ①B: 340.5 ng/ul 1.84; ③A: 116.2 ng/ul 1.65; ③B: 102.1 ng/ul 1.77; ⑤A: 151.4 ng/ul 1.80; ⑤B: 68.0 ng/ul 1.84;

7.25

Transformation of ②,④,⑥,⑦,⑧,⑨

Time: 0:00

Handler: Tang Shiqiang

Procedure: IGEM Protocols

Picking single colonies of ②,④,⑥,⑦,⑧,⑨

Time: 20:00

Handler: Tang Shiqiang

Procedure:

  1. Pick single colonies, adding 6ml LB with corresponding antibiotics, each plate we pick 2 single colonies, α and β.
  2. Incubate overnight at 37℃, 220rpm.

7.26

Plasmid extraction of ②,④,⑥,⑦,⑧,⑨ group α

Time: 9:40

Handler: Lu Shixin

Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol

Results:

Concentration: ②α: 369.8 ng/ul; ④α: 142.4 ng/ul; ⑥α: 116.5 ng/ul; ⑦α: 92 ng/ul; ⑧α: 157.4 ng/ul; ⑨α: 168.3 ng/ul.

‘’’ Note: ’’’ ⑧ has a red colony.

Plasmid extraction of ②,④,⑥,⑦,⑧,⑨ group β

Time: 14:00

Handler: Tang Shiqiang

Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol

Results:

Concentration: ②β: 496.9 ng/ul 1.86; ④β: 256.4 ng/ul 1.85; ⑥β: 220.3 ng/ul 1.85; ⑦β: 225.7 ng/ul 1.85; ⑧β: 304.6 ng/ul 1.85; ⑨β: 352.2 ng/ul 1.84.

‘’’ Note: ‘’’ ⑧ has a red colony.

7.27

Competent Cell Test

Time: 16:00

Handler: Lu Shixin

Procedure: IGEM Protocols

  1. Spin down the DNA tubes form the Competent Cell Test Kit to collect all of the DNA into the bottom of each tube prior to use.
  2. Thaw competent cells on ice.
  3. Pipet 50ul of competent cells into each tube.
  4. Pipet 1ul of DNA into each microcentrifuge tube.
  5. Incubate on ice for 30 min.
  6. Heat shock at 42℃ for 1 min.
  7. Incubate on ice for 5 min.
  8. Add 200ul SOC media, incubate at 37℃ for 2h.
  9. Pipet 20ul from each tube onto the appropriate plate, and spread it.
  10. Incubate at 37℃ overnight, approximately 16 h.

Enzyme Digestion and Go Gel Electrophoresis Test of ②,④,⑥,⑦,⑧,⑨

Time: 20:00

Handler: Xiao Tianyao

Procedure:

1. Make enzyme cutting system solution.

②α ④α ⑥α ⑦α ⑧α ⑨α
DNA(400ng) 1ul 2ul 2ul 2ul 1.2ul 1.2ul
Cutsmart buffer 2ul 2ul 2ul 2ul 2ul 2ul
EcoRI-HF 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul
PstI-HF 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul
ddH2O 16.6ul 15.6ul 15.6ul 15.6ul 16.4ul 16.4ul
Total 20ul 20ul 20ul 20ul 20ul 20ul

②β ④β ⑥β ⑦β ⑧β ⑨β
DNA(400ng) 1ul 2ul 2ul 2ul 1.2ul 1.2ul
Cutsmart buffer 2ul 2ul 2ul 2ul 2ul 2ul
EcoRI-HF 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul
PstI-HF 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul
ddH2O 16.6ul 15.6ul 15.6ul 15.6ul 16.4ul 16.4ul
Total 20ul 20ul 20ul 20ul 20ul 20ul


  1. Incubate at 37℃ for 30 min.

  2. Make 1% agarose gel 30ml.

  3. Add 6x loading dye to the enzyme cutting system solution.

  4. Go gel electrophoresis.

Results:

figures show below:

Comment: ⑧ has some problem, and we will redo it.

7.28

Competent Cell Test Colonies Counting

Time: 13:00

Handler: Lu Shixin

Result:

Figure 1. Water control
Figure 2. 0.5 pg/ul
Figure 3. 5 pg/ul
Figure 4. 10 pg/ul
Figure 5. 20 pg/ul

[[File:SUSTech_Shenzhen-B9DEB8C5-5470-42AE-A894-F75F70DEE777.png | 200px | thumb | left | Figure 6. 50 pg/ul]

SUSTech Shenzhen-372E5718-44FE-463A-86A8-57C609A8F345.png

And all the colonies are red under UV light.

7.29

Transformation of ⑧,⑩，11

Time: 16:00

Handler: Lu Shixin, Tang Shiqiang

Procedure: IGEM Transformation Protocol

Enzyme Digestion for Gel Extraction of ⑤,⑥,⑦,⑧,⑨

Time: 20:00

Handler: LuShixin, Tang Shiqiang

Procedure:

  1. Make enzyme cutting system solution.
⑥ ⑦ ⑧ ⑨
DNA（500ng） 2.5ul 2.5ul 2.0ul 1.5ul
Cutsmart buffer 2.5ul 2.5ul 2.5ul 2.5ul
EcoRI-HF 0.5ul 0.5ul 0.5ul 0.5ul
SpeI-HF 0.5ul 0.5ul 0.5ul 0.5ul
ddH2O 19ul 19ul 19.5ul 20ul
Total 25ul 25ul 25ul 25ul
⑤
DNA (500ng) 8ul
Cutsmart buffer 2.5ul
EcoRI-HF 0.5ul
XbaI 0.5ul
ddH2O 16.5ul
Total 25ul
  1. Incubate at 37℃ overnight. (Start from 21:20)

7.30

Picking single colonies of ⑧, ⑩, 11

Time: 9:00

Handler: Lu Shixin

Procedure:

  1. Pick single colonies, adding 1ml LB with corresponding antibiotics in 1.5ml EP tube, each plate we pick 2 single colonies, α and β.
  2. Incubate for 2h at 37℃.
  3. Transfer 1ml LB from 1.5ml EP tube to 14ml bacteria shaking tube, and add 5ml corresponding antibiotics LB.
  4. Incubate at 37℃ for 10h, 220rpm.

Go Gel Electrophoresis and Gel Extraction of ⑤,⑥,⑦,⑧,⑨

Time: 9:00

Handler: Lu Shixin

Procedure: bio-tek OMEGA E.Z.N.A. Gel Extraction Kit – Spin Protocol

  1. Make 1% agarose gel 30ml.
  2. Add 6x loading dye to the enzyme cutting system solution.
  3. Go gel electrophoresis.
  4. Excise the DNA fragment of interest.
  5. Determine the volume of the gel slice by weighing it in a clean 1.5ml EP tube.
  6. Add 1 volume Binding Buffer (XP2).
  7. Incubate at 50-60℃ for 7 min or until the gel has completely melted. Vortex or shake the tube every 2-3 min.
  8. Insert a HiBind DNA Mini Column in a 2ml Collection Tube.
  9. Add no more than 700ul DNA/agarose solution from Step 7 to the HiBind DNA Mini Column.
  10. Centrifuge at 10,000 g for 1 min at room temperature.
  11. Discard the filtrate and reuse collection tube.
  12. Repeat Steps 9-11 until all of the sample has been transferred to the column.
  13. Add 300ul Binding Buffer(XP2).
  14. Centrifuge at maximum speed (>13,000 g) for 1 min at room temperature.
  15. Discard the filtrate and reuse collection tube.
  16. Add 700ul SPW Wash Buffer. (SPW Wash Buffer must be diluted with 100% ethanol prior to use.)
  17. Centrifuge at maximum speed for 1 min at room temperature.
  18. Discard the filtrate and reuse collection tube.
  19. Repeat Steps 16-18.
  20. Centrifuge the empty HiBind DNA Mini Column for 2 min at maximum speed to dry the column matrix.
  21. Transfer the HiBind DNA Mini Column to a clean 1.5ml EP tube.
  22. Add 15-30ul ddH2O.
  23. Let sit at room temperature for 2 min.
  24. Centrifuge at maximum speed for 1 min.

Results：

SUSTech Shenzhen-4E6C74F1-3B4C-4F1A-B283-6751068590D8.png

All the concentration of gel extraction are below 10 ng/ul. So we decide to repeat enzyme digestion with more DNA.

Enzyme Digestion for Gel Extraction of ⑤,⑥,⑦,⑨ (Redo)

Time: 18:00

Handler: Lu Shixin

Procedure:

  1. Make enzyme cutting system solution.
⑥ ⑦ ⑨
DNA（>4ug） 21.5ul 21.5ul 21.5ul
Cutsmart buffer 2.5ul 2.5ul 2.5ul
EcoRI-HF 0.5ul 0.5ul 0.5ul
SpeI-HF 0.5ul 0.5ul 0.5ul
ddH2O 0ul 0ul 0ul
Total 25ul 25ul 25ul
⑤
DNA (2ug) 21.5ul
Cutsmart buffer 2.5ul
EcoRI-HF 0.5ul
XbaI 0.5ul
ddH2O 0ul
Total 25ul
  1. Incubate at 37℃ for 3h. (Start from 18:40)

Plasmid extraction of ⑧,⑩,11

Time: 21:00

Handler: Lu Shixin

Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol

Results:

The length of them are correct.

Concentration: ⑧: 130.2 ng/ul; ⑩: 147.0 ng/ul; 11: 93.3 ng/ul.

Enzyme Digestion for Gel Extraction of ⑧,⑩,11

Time: 22:30

Handler: Lu Shixin

Procedure:

  1. Make enzyme cutting system solution.
⑩ 11
DNA（>1.5ug） 11.5ul 21.5ul
Cutsmart buffer 2.5ul 2.5ul
SpeI-HF 0.5ul 0.5ul
PstI-HF 0.5ul 0.5ul
ddH2O 10ul 0ul
Total 25ul 25ul
⑧
DNA (0.5ug) 4ul
Cutsmart buffer 2.5ul
EcoRI-HF 0.5ul
XbaI 0.5ul
ddH2O 17.5ul
Total 25ul
  1. Incubate at 37℃ overnight. (Start from 22:40)

Go Gel Electrophoresis and Gel Extraction of ⑤,⑥,⑦,⑨ (Redo)

Time: 22:00

Handler: Tang Shiqiang

Procedure: bio-tek OMEGA E.Z.N.A. Gel Extraction Kit – Spin Protocol

Results:

Gel extraction concentration: ⑤: 15.3 ng/ul; ⑥: 36.3 ng/ul; ⑦: 36.8 ng/ul; ⑨: 26.9 ng/ul.

7.31

Ligation of ⑥+⑤, ⑦+⑤, ⑨+⑤

Time: 0:00

Handler: Tang Shiqiang

Procedure:

  1. Make ligation system solution.
⑥+⑤ ⑦+⑤ ⑨+⑤
Vector (50ng) 3.3ul 3.3ul 3.3ul
Insert 1.4ul 1.4ul 1.9ul
ddH2O 5.3ul 5.3ul 4.8ul
2x Quick ligase buffer 10ul 10ul 10ul
Quick ligase 1ul 1ul 1ul
Total 21ul 21ul 21ul
  1. Incubate at room temperature for 5 min.
  2. Take 2.5ul ligation system solution to do transformation.

Go Gel Electrophoresis and Gel Extraction of ⑧, ⑩, 11

Time: 15:30

Handler: Lu Shixin

Procedure: bio-tek OMEGA E.Z.N.A. Gel Extraction Kit – Spin Protocol

Results:

SUSTech Shenzhen-FF23D23B-B68F-42EB-87A2-DBDED38BDF8E.png

Gel extraction concentration: ⑩: 67.3 ng/ul 1.65; 11: 66.4 ng/ul 1.69.

‘’‘Note:’‘’ We found that we take a wrong ⑧ before.

Transformation of ⑧

Time: 16:30

Handler: Tang Shiqiang

Procedure: IGEM Transformation Protocol

Picking single colonies of ⑥+⑤, ⑦+⑤, ⑨+⑤

Time: 19:30

Handler: Tang Shiqiang

Procedure:

  1. Pick single colonies, adding 6ml LB with corresponding antibiotics, each plate we pick 2 single colonies, A and B.
  2. Incubate overnight at 37℃, 220rpm.

8.1

Picking single colonies of ⑧

Time: 9:30

Handler: Tang Shiqiang

Procedure:

  1. Pick single colonies, adding 6ml LB with corresponding antibiotics, each plate we pick 2 single colonies, A and B.
  2. Incubate at 37℃ for about 14h, 220rpm.

Plasmid extraction of ⑥+⑤, ⑦+⑤, ⑨+⑤ A and B

Time: 9:30

Handler: Tang Shiqiang

Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol

Results:

Concentration: ⑥+⑤A: 126.3 ng/ul 1.86; ⑥+⑤B: 124.9 ng/ul 1.82; ⑦+⑤A: 116.5 ng/ul 1.87; ⑦+⑤B: 112.0 ng/ul 1.86; ⑨+⑤A: 120.4 ng/ul 1.87; ⑨+⑤B: 149.0 ng/ul 1.87;

Enzyme Digestion and Go Gel Electrophoresis Test of ⑥+⑤, ⑦+⑤, ⑨+⑤ A and B

Time: 12:00

Handler: Tang Shiqiang

Procedure:

  1. Make enzyme cutting system solution.
⑥+⑤A ⑦+⑤A ⑨+⑤A ⑥+⑤B ⑦+⑤B ⑨+⑤B
DNA(200ng) 2ul 2ul 2ul 2ul 2ul 2ul
Cutsmart buffer 2ul 2ul 2ul 2ul 2ul 2ul
EcoRI-HF 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul
PstI-HF 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul
ddH2O 15.6ul 15.6ul 15.6ul 15.6ul 15.6ul 15.6ul
Total 20ul 20ul 20ul 20ul 20ul 20ul
  1. Incubate at 37℃ for 30 min.
  2. Make 1% agarose gel 30ml.
  3. Add 6x loading dye to the enzyme cutting system solution.
  4. Go gel electrophoresis.

Results:

SUSTech Shenzhen-3DD57F02-827C-43BC-B57F-2ACCA230ECF4.png

‘’‘Comments:’‘’ The length of them are incorrect. Their length are larger than 1000bp while should be smaller than 1000bp.

‘’‘Note:’‘’ Finally we found that we used a tube of wrong ⑤.

Put ⑧ A and B at 4℃

Time: 20:00

Handler: Tang Shiqiang

8.2

Plasmid extraction of ⑧ A and B

Time: 14:00

Handler: Tang Shiqiang

Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol

Results:

Concentration: ⑧A: 116.5 ng/ul 1.82; ⑧B: 73.2 ng/ul 1.81.

Enzyme Digestion for Gel Extraction of ⑤, ⑧

Time: 22:00

Handler: Lu Shixin, Tang Shiqiang

Procedure:

  1. Make enzyme cutting system solution.
⑤3-3-F 1
DNA（>3ug） 21.5ul
Cutsmart buffer 2.5ul
EcoRI-HF 0.5ul
XbaI 0.5ul
ddH2O 0ul
Total 25ul
⑧A
DNA (>2ug) 21.5ul
Cutsmart buffer 2.5ul
EcoRI-HF 0.5ul
SpeI-HF 0.5ul
ddH2O 0ul
Total 25ul
  1. Incubate at 37℃ overnight. (Start from 22:25)

8.3

Go Gel Electrophoresis and Gel Extraction of ⑤, ⑧

Time: 10:00

Handler: Lu Shixin

Procedure: bio-tek OMEGA E.Z.N.A. Gel Extraction Kit – Spin Protocol

Results:

SUSTech Shenzhen-0D26F48D-11BF-4C72-B460-51AB3110125F.png

Concentration: ⑤: 51.4 ng/ul 1.75; ⑧: 23.2 ng/ul 1.71.

Ligation of ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤

Time: 16:30

Handler: Lu Shixin

Procedure:

  1. Make ligation system solution.
⑥+⑤ ⑦+⑤ ⑧+⑤ ⑨+⑤
Vector (50ng) 1ul 1ul 1ul 1ul
Insert 1.5ul 1.5ul 2.5ul 2ul
ddH2O 7.5ul 7.5ul 6.5ul 7ul
2x Quick ligase buffer 10ul 10ul 10ul 10ul
Quick ligase 1ul 1ul 1ul 1ul
Total 21ul 21ul 21ul 21ul
  1. Incubate at room temperature for 10 min.
  2. Take 2ul ligation system solution to do transformation.

8.4

Picking single colonies of ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤

Time: 9:30

Handler: Lu Shixin

Procedure:

  1. Pick single colonies, adding 6ml LB with corresponding antibiotics, each plate we pick 2 single colonies, A and B.
  2. Incubate at 37℃ for about 12h, 220rpm.

Plasmid extraction of ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤ A and B

Time: 21:30

Handler: Tang Shiqiang

Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol

Results:

Concentration: ⑥+⑤A: 54.4 ng/ul 2.18; ⑥+⑤B: 29.2 ng/ul 3.15; ⑦+⑤A: 55.4 ng/ul 2.42; ⑦+⑤B: 58.8 ng/ul 2.42; ⑧+⑤A: 70.0 ng/ul 2.22; ⑧+⑤B: 35.6 ng/ul 2.84; ⑨+⑤A: 65.9 ng/ul 2.23; ⑨+⑤B: 74.3 ng/ul 2.18;

‘’’Comments:’’’ All the concentration seems to have some problem, and the A260/280 have some problem.

Enzyme Digestion and Go Gel Electrophoresis Test of ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤ A and B

Time: 23:00

Handler: Tang Shiqiang

Procedure:

  1. Make enzyme cutting system solution.
⑥+⑤A ⑦+⑤A ⑧+⑤A ⑨+⑤A ⑥+⑤B ⑦+⑤B ⑧+⑤B ⑨+⑤B
DNA(200ng) 4ul 4ul 3ul 3ul 7ul 4ul 7ul 3ul
Cutsmart buffer 2ul 2ul 2ul 2ul 2ul 2ul 2ul 2ul
XbaI 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul
PstI-HF 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul
ddH2O 13.6ul 13.6ul 14.6ul 14.6ul 10.6ul 13.6ul 10.6ul 14.6ul
Total 20ul 20ul 20ul 20ul 20ul 20ul 20ul 20ul
  1. Incubate at 37℃ for 30 min.
  2. Make 1% agarose gel 30ml.
  3. Add 6x loading dye to the enzyme cutting system solution.
  4. Go gel electrophoresis.

Results:

The length seems to be correct.

8.5

Enzyme Digestion for Gel Extraction of ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤

Time: 2:20

Handler: Tang Shiqiang

Procedure:

  1. Make enzyme cutting system solution.
⑥+⑤A ⑦+⑤B ⑧+⑤A ⑨+⑤B
DNA（>1.5ug） 26ul 26ul 26ul 26ul
Cutsmart buffer 3ul 3ul 3ul 3ul
EcoRI-HF 0.5ul 0.5ul 0.5ul 0.5ul
XbaI 0.5ul 0.5ul 0.5ul 0.5ul
ddH2O 0ul 0ul 0ul 0ul
Total 30ul 30ul 30ul 30ul

  1. Incubate at 37℃ overnight. (Start from 2:30)

Go Gel Electrophoresis and Gel Extraction of ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤

Time: 8:10

Handler: Lu Shixin, Tang Shiqiang

Procedure: bio-tek OMEGA E.Z.N.A. Gel Extraction Kit – Spin Protocol

Results:

Concentration: ⑥+⑤: 25.1 ng/ul 1.58; ⑦+⑤: 23.4 ng/ul 1.54; ⑧+⑤: 45.9 ng/ul 1.01; ⑨+⑤: 26.7 ng/ul 1.50;

Ligation of ⑩,11 and ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤

Time: 11:00

Handler: Lu Shixin, Tang Shiqiang

Procedure:

  1. Make ligation system solution.
⑩+⑥+⑤ ⑩+⑦+⑤ ⑩+⑧+⑤ ⑩+⑨+⑤
Vector ⑩ (50ng) 0.8ul 0.8ul 0.8ul 0.8ul
Insert 3.4ul 3.4ul 2ul 3.4ul
ddH2O 5.8ul 5.8ul 7.2ul 5.8ul
2x Quick ligase buffer 10ul 10ul 10ul 10ul
Quick ligase 1ul 1ul 1ul 1ul
Total 21ul 21ul 21ul 21ul
11+⑥+⑤ 11+⑦+⑤ 11+⑧+⑤ 11+⑨+⑤
Vector ⑩ (50ng) 0.8ul 0.8ul 0.8ul 0.8ul
Insert 3.4ul 3.4ul 2ul 3.4ul
ddH2O 5.8ul 5.8ul 7.2ul 5.8ul
2x Quick ligase buffer 10ul 10ul 10ul 10ul
Quick ligase 1ul 1ul 1ul 1ul
Total 21ul 21ul 21ul 21ul
  1. Incubate at room temperature for 10 min.
  2. Take 5ul ligation system solution to do transformation.

8.6

Observation of single colonies of ⑩+⑥+⑤, ⑩+⑦+⑤, ⑩+⑧+⑤, ⑩+⑨+⑤, 11+⑥+⑤, 11+⑦+⑤, 11+⑧+⑤, 11+⑨+⑤

Time: 7:00

Handler: Xiao Tianyao

Results:

Colonies number
⑩ backbone control 5
11 backbone control 20
⑩+⑥+⑤ None
⑩+⑦+⑤ 1
⑩+⑧+⑤ 20
⑩+⑨+⑤ >50
11+⑥+⑤ 4
11+⑦+⑤ 1
11+⑧+⑤ 6
11+⑨+⑤ >50

Comments: Control groups have colonies. So the ligation results are improbable. We would redo it.

Picking single colonies of ⑩+⑦+⑤, ⑩+⑧+⑤, ⑩+⑨+⑤, 11+⑥+⑤, 11+⑦+⑤, 11+⑧+⑤, 11+⑨+⑤

Time: 10:00

Handler: Liao Weiduo

Procedure:

  1. Pick single colonies, adding 3 ml C+ LB.
  2. Incubate at 37℃ for about 12h, 220rpm.

Picking single colonies of ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤

Time: 11:00

Handler: Tang Shiqiang

Procedure:

  1. Pick single colonies, adding 6ml LB with corresponding antibiotics, each plate we pick 2 single colonies, C and D.
  2. Incubate at 37℃ for about 12h, 220rpm.

Plasmid extraction of ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤ A and B

Time: 23:00

Handler: Tang Shiqiang

Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol

Results:

Concentration: ⑥+⑤C: 63.8 ng/ul 1.81; ⑥+⑤D: 59.4 ng/ul 1.79; ⑦+⑤C: 49.9 ng/ul 1.81; ⑦+⑤D: 65.8 ng/ul 1.75; ⑧+⑤C: 73.4ng/ul 1.81; ⑧+⑤D: 72.8 ng/ul 1.81; ⑨+⑤C: 65.8 ng/ul 1.74; ⑨+⑤D: 64.1 ng/ul 1.83.

8.7

Enzyme Digestion and Go Gel Electrophoresis Test of ⑥+⑤, ⑦+⑤, ⑧+⑤, ⑨+⑤ C and D

Time: 2:00

Handler: Tang Shiqiang

Procedure:

  1. Make enzyme cutting system solution.
⑥+⑤C ⑦+⑤C ⑧+⑤C ⑨+⑤C ⑥+⑤D ⑦+⑤D ⑧+⑤D ⑨+⑤D
DNA(100ng) 1.5ul 1.5ul 1.5ul 1.5ul 1.5ul 1.5ul 1.5ul 1.5ul
Cutsmart buffer 2ul 2ul 2ul 2ul 2ul 2ul 2ul 2ul
XbaI 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul
PstI-HF 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul
ddH2O 16.1ul 16.1ul 16.1ul 16.1ul 16.1ul 16.1ul 16.1ul 1.16ul
Total 20ul 20ul 20ul 20ul 20ul 20ul 20ul 20ul
  1. Incubate at 37℃ for 30 min.
  2. Make 1% agarose gel 30ml.
  3. Add 6x loading dye to the enzyme cutting system solution.
  4. Go gel electrophoresis.

Results:

SUSTech Shenzhen-1C4A2A62-E9CB-49BE-B41A-544A82C84E24.png

Observation of LB medium color of ⑩+⑥+⑤, ⑩+⑦+⑤, ⑩+⑧+⑤, ⑩+⑨+⑤, 11+⑥+⑤, 11+⑦+⑤, 11+⑧+⑤, 11+⑨+⑤

Time: 7:00

Handler: Xiao Tianyao

Results:

Color
⑩+⑥+⑤ None
⑩+⑦+⑤ Purple
⑩+⑧+⑤ Yellow
⑩+⑨+⑤ Red
11+⑥+⑤ Red
11+⑦+⑤ None
11+⑧+⑤ None
11+⑨+⑤ None

‘’’Note:’’’ There are yellow colonies on plate 11+⑧+⑤ and red colonies on 11+⑨+⑤.

Picking single colonies of ⑩+⑦+⑤, ⑩+⑧+⑤, ⑩+⑨+⑤, 11+⑥+⑤, 11+⑧+⑤, 11+⑨+⑤ （Because there are obvious correct color colonies on those plates）

Time: 9:30

Handler: Liao Weiduo

Procedure:

  1. Pick single colonies, adding 6 ml C+ LB.
  2. Incubate at 37℃ for about 12h, 220rpm.

Ligation of ⑩ and ⑥+⑤, 11 and ⑦+⑤

Time: 11:00

Handler: Tang Shiqiang

Procedure:

  1. Make ligation system solution.
⑩+⑥+⑤
Vector ⑩ (50ng) 0.7ul
Insert 3.2ul
ddH2O 6.1ul
2x Quick ligase buffer 10ul
Quick ligase 1ul
Total 21ul
11+⑦+⑤
Vector ⑩ (50ng) 0.7ul
Insert 3.2ul
ddH2O 6.1ul
2x Quick ligase buffer 10ul
Quick ligase 1ul
Total 21ul
  1. Incubate at room temperature for 10 min.
  2. Take 5ul ligation system solution to do transformation.

Plasmid extraction of ⑩+⑦+⑤, ⑩+⑧+⑤, ⑩+⑨+⑤, 11+⑥+⑤, 11+⑧+⑤, 11+⑨+⑤

Time: 22:00

Handler: Liao Weiduo

Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol

Results:

Concentration: ⑩+⑦+⑤: 63.6 ng/ul 1.71; ⑩+⑧+⑤: 100.7 ng/ul 1.54; ⑩+⑨+⑤: 83.0 ng/ul 1.59; 11+⑥+⑤: 114.8 ng/ul 1.65; 11+⑧+⑤: 77.5 ng/ul 1.78; 11+⑨+⑤: 106.3 ng/ul 1.51.

8.9

Picking single colonies of ⑩+⑥+⑤, 11+⑦+⑤

Time: 00:00

Handler: Tang Shiqiang

Procedure:

  1. Pick single colonies, adding 6ml LB with corresponding antibiotics.
  2. Incubate overnight at 37℃, 220rpm.

Plasmid extraction of ⑩+⑥+⑤, 11+⑦+⑤

Time: 16:00

Handler: Tang Shiqiang

Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol

Results:

Concentration: ⑩+⑥+⑤: 109.0 ng/ul 1.74; 11+⑦+⑤: 115.3 ng/ul 1.85.

sfGFG

8.2

Plate streaking of a plasmid with contain sfGFP and msfGFP

Time: 17:30

Handler: Tang Shiqiang

8.3

Picking single colonies of sfGFP and msfGFP

Time: 9:00

Handler: Tang Shiqiang

Procedure:

  1. Pick single colonies, adding 6ml LB with corresponding antibiotics.
  2. Incubate for about 12h at 37℃, 220rpm.

Plasmid extraction of sfGFP and msfGFP

Time: 21:30

Handler: Lu Shixin

Procedure:bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol

Result:

Concentration: sfGFP: 70.0 ng/ul 1.77; msfGFP: 460.8 ng/ul.

8.16

PCR of sfGFP and msfGFP

Time: 16:00

Handler: Tang Shiqiang

Procedure:

DNA 0.1ul
bbp3 2.5ul
bbp4 2.5ul
Q5 25ul
ddH2O 20ul
Total 50ul
32 cycles Temperature Time
Initialization 98℃ 30s
Denaturation 98℃ 5s
Annealing 65℃ 20s
Extension 72℃ 10s
Final elongation 72℃ 2min

Go Gel Electrophoresis and Gel Extraction of sfGFP and msfGFP PCR product

Time: 19:00

Handler: Tang Shiqiang

Procedure: bio-tek OMEGA E.Z.N.A. Gel Extraction Kit – Spin Protocol

Result:

Concentration: sfGFP: 43.2 ng/ul 1.76; msfGFP: 62.0 ng/ul 1.79.

Enzyme Digestion for Gel Extraction of sfGFP and msfGFP

Time: 19:30

Handler: Tang Shiqiang

Procedure:

  1. Make enzyme cutting system solution.
sfGFP msfGFP
DNA（>1ug） 27.5ul 27.5ul
Cutsmart buffer 2.5ul 2.5ul
EcoRI-HF 0.5ul 0.5ul
SpeI-HF 0.5ul 0.5ul
ddH2O 19ul 19ul
Total 50ul 50ul
  1. Incubate at 37℃ for 3h.

Enzyme Digestion for Gel Extraction of ⑤

Time: 17:00

Handler: Tang Shiqiang

Procedure:

  1. Make enzyme cutting system solution.
⑤
DNA（>3ug） 21.5ul
Cutsmart buffer 2.5ul
EcoRI-HF 0.5ul
SpeI-HF 0.5ul
ddH2O 0
Total 25ul
  1. Incubate at 37℃ for 3h

Go Gel Electrophoresis and Gel Extraction of sfGFP, msfGFP, and ⑤

Time: 20:00

Handler: Tang Shiqiang

Procedure: bio-tek OMEGA E.Z.N.A. Gel Extraction Kit – Spin Protocol

Results:

SUSTech Shenzhen-4E05620C-3E12-4366-BF74-FE6EC5D89D63.png

Gel extraction concentration: ⑤: 61.4 ng/ul 1.58; sfGFP: 16.9 ng/ul 1.62; msfGFP: 16.5 ng/ul 1.59.

8.17

Ligation of sfGFP+⑤, msfGFP+⑤, sfGFP+pSB1C3, msfGFP+pSB1C3

Time: 0:00

Handler: Tang Shiqiang

Procedure:

  1. Make ligation system solution.
sfGFP msfGFP
Vector ⑤ (50ng) 1ul 1ul
Insert 4ul 4ul
ddH2O 0ul 0ul
2x Quick ligase buffer 5ul 5ul
Quick ligase 0.5ul 0.5ul
Total 10.5ul 10.5ul
  1. Incubate at room temperature for 10 min.
  2. Take 10.5ul ligation system solution to do transformation.

Picking single colonies of sfGFP and msfGFP

Time: 18:30

Handler: Tang Shiqiang

Procedure:

  1. Pick single colonies, adding 6ml LB with corresponding antibiotics.
  2. Incubate overnight at 37℃, 220rpm.

8.18

Plasmid extraction of sfGFP+⑤, msfGFP+⑤, sfGFP+pSB1C3, msfGFP+pSB1C3

Time: 13:00

Handler: Tang Shiqiang

Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol

Result:

Concentration: sfGFP+⑤: 103.6 ng/ul 1.96; msfGFP+⑤: 127.1 ng/ul 1.80; sfGFP+pSB1C3①: 156.1 ng/ul 1.91; sfGFP+pSB1C3②: 179.2 ng/ul 1.91; msfGFP+pSB1C3①: 115.5 ng/ul 1.93; msfGFP+pSB1C3②: 159.5 ng/ul 1.92.

8.18

Enzyme Digestion and Go Gel Electrophoresis Test of sfGFP+⑤, msfGFP+⑤, sfGFP+pSB1C3, msfGFP+pSB1C3

Time: 0:00

Handler: Tang Shiqiang

Procedure:

  1. Make enzyme cutting system solution.
sfGFP+⑤ msfGFP+⑤ sfGFP+pSB1C3① sfGFP+pSB1C3② msfGFP+pSB1C3① msfGFP+pSB1C3②
DNA(200ng) 2ul 2ul 2ul 2ul 2ul 2ul
Cutsmart buffer 2ul 2ul 2ul 2ul 2ul 2ul
KpnI-HF 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul
ddH2O 16.8ul 16.8ul 16.8ul 16.8ul 16.8ul 16.8ul
Total 20ul 20ul 20ul 20ul 20ul 20ul
sfGFP+⑤ msfGFP+⑤ sfGFP+pSB1C3① sfGFP+pSB1C3② msfGFP+pSB1C3① msfGFP+pSB1C3②
DNA(200ng) 2ul 2ul 2ul 2ul 2ul 2ul
Cutsmart buffer 2ul 2ul 2ul 2ul 2ul 2ul
EcoRI-HF 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul
ddH2O 16.8ul 16.8ul 16.8ul 16.8ul 16.8ul 16.8ul
Total 20ul 20ul 20ul 20ul 20ul 20ul
sfGFP+⑤ msfGFP+⑤ sfGFP+pSB1C3① sfGFP+pSB1C3② msfGFP+pSB1C3① msfGFP+pSB1C3②
DNA(200ng) 2ul 2ul 2ul 2ul 2ul 2ul
Cutsmart buffer 2ul 2ul 2ul 2ul 2ul 2ul
EcoRI-HF 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul
PstI-HF 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul
ddH2O 16.6ul 16.6ul 16.6ul 16.6ul 16.6ul 16.6ul
Total 20ul 20ul 20ul 20ul 20ul 20ul
  1. Incubate overnight at 37℃.
  2. Make 1% agarose gel 30ml.
  3. Add 6x loading dye to the enzyme cutting system solution.
  4. Go gel electrophoresis.

Results:

SUSTech Shenzhen-CEC5E686-4611-48DE-97D5-7B98E16B8453.png

9.21

PCR of msfGFP Coding sequence basic part

Time: 16:00

Handler: Tang Shiqiang

Procedure:

DNA 1ul
SfGFP-forward 1ul
bbp4 1ul
primeSTAR 20ul
ddH2O 17ul
Total 40ul
32 cycles Temperature Time
Initialization 98℃ 30s
Denaturation 98℃ 10s
Annealing 65℃ 5s
Extension 72℃ 5s
Final elongation 72℃ 2min

Go Gel Electrophoresis and Gel Extraction of msfGFP CDS

Time: 20:00

Handler: Tang Shiqiang

Procedure: bio-tek OMEGA E.Z.N.A. Gel Extraction Kit – Spin Protocol

Result:

Concentration: msfGFP: 96.1 ng/ul 1.83.

Enzyme Digestion for Gel Extraction of msfGFP CDS

Time: 20:30

Handler: Tang Shiqiang

Procedure:

  1. Make enzyme cutting system solution.
msfGFP CDS
DNA 43ul
Cutsmart buffer 5ul
EcoRI-HF 1ul
SpeI-HF 1ul
ddH2O 0ul
Total 50ul
  1. Incubate overnight at 37℃.

9.22

Transformation of msfGFP CDS

Time: 11:00

Handler: Tang Shiqiang

Procedure: IGEM Protocols

Picking single colonies of sfGFP CDS

Time: 23:00

Handler: Tang Shiqiang

Procedure:

  1. Pick single colonies, adding 5ml C+ LB.
  2. Incubate overnight at 37℃, 220rpm.

9.23

Plasmid extraction of msfGFP CDS

Time: 17:00

Handler: Tang Shiqiang

Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol

Result:

Concentration: msfGFP CDS①: 141.0 ng/ul 1.88; msfGFP CDS②: 141.0 ng/ul 1.90.

9.24

Enzyme Digestion and Go Gel Electrophoresis Test of msfGFP CDS① and msfGFP CDS②

Time: 11:40

Handler: Tang Shiqiang

Procedure:

  1. Make enzyme cutting system solution.
msfGFP CDS① msfGFP CDS②
DNA(300ng) 2.5ul 2.5ul
Cutsmart buffer 1ul 1ul
MluI-HF/MscI 0.5ul 0.5ul
ddH2O 6ul 6ul
Total 10ul 10ul
  1. Incubate for 3h at 37℃.
  2. Make 1% agarose gel 30ml.
  3. Add 6x loading dye to the enzyme cutting system solution.
  4. Go gel electrophoresis.

Results:

SUSTech Shenzhen-C3B1C094-5150-4A74-A235-7F3C30D4AD67.png

eukaryon antibiotic

8.31

PCR of bla, bleo, puro, zeo

Time: 16:00

Handler: Tang Shiqiang

Procedure:

DNA 0.5ul
Forward 1.25ul
Reverse 1.25ul
PrimeSTAR 25ul
ddH2O 22ul
Total 50ul
33 cycles Temperature Time
Initialization 98℃ 30s
Denaturation 98℃ 10s
Annealing 55℃ 15s
Extension 72℃ 5s
Final elongation 72℃ 2min

Go Gel Electrophoresis and Gel Extraction of bla, bleo, puro, zeo PCR product

Time: 18:00

Handler: Tang Shiqiang

Procedure: bio-tek OMEGA E.Z.N.A. Gel Extraction Kit – Spin Protocol

Result:

SUSTech Shenzhen-B708EBD5-22F3-4C11-B5E6-DDDFD8A55079.png

Concentration: bla: 66.6 ng/ul 1.91; bleo: 41.2 ng/ul 1.88; puro: 52.7 ng/ul 1.99; zeo: 46.3 ng/ul 1.97.

Enzyme Digestion for Gel Extraction of bla, bleo, puro, zeo

Time: 19:30

Handler: Tang Shiqiang

Procedure:

  1. Make enzyme cutting system solution.
bla bleo puro zeo
DNA（>1ug） 28ul 28ul 28ul 28ul
Cutsmart buffer 5ul 5ul 5ul 5ul
EcoRI-HF 1ul 1ul 1ul 1ul
SpeI-HF 1ul 1ul 1ul 1ul
ddH2O 15ul 15ul 15ul 15ul
Total 50ul 50ul 50ul 50ul
  1. Incubate at 37℃ for 4h.

Cycle Pure of bla, bleo, puro, zeo

Time: 23:30

Handler: Tang Shiqiang

Procedure: bio-tek OMEGA E.Z.N.A. Cycle Pure Kit Centrifugation Protocol

  1. Transfer the PCR sample into a clean 1.5ml microcentrifuge tube.
  2. Add 4-5 volumes CP Buffer.
  3. Vortex to mix thoroughly.
  4. Insert a HiBind DNA Mini Column into a 2ml Collection Tube.
  5. Add the sample from Step 3 to the HiBind DNA Min Column.
  6. Centrifuge at maximum speed (>13,000 g) for 1 min at room temperature.
  7. Discard the filtrate and reuse collection tube.
  8. Add 700ul DNA Wash Buffer.
  9. Centrifuge at maximum speed for 1 min.
  10. Discard the filtrate and reuse collection tube.
  11. Repeat Step 8-10.
  12. Centrifuge the empty HiBind DNA Mini Column at maximum speed for 2 min.
  13. Transfer the HiBind DNA Mini Column into a clean 1.5ml microcentrifuge tube.
  14. Add 30-50ul ddH2O.
  15. Let sit at room temperature for 2 min.
  16. Centrifuge at maximum speed for 1 min.

Result:

Concentration: bla: 46.2 ng/ul 1.74; bleo: 20.3 ng/ul 1.32; puro: 32.4 ng/ul 1.50; zeo: 32.4 ng/ul 1.76.

9.2

Ligation of bla, bleo, puro, zeo to pSB1C3

Time: 20:30

Handler: Liao Weiduo

Procedure:

  1. Make ligation system solution.
bla bleo puro zeo
Vector(50ng) 2ul 2ul 2ul 2ul
Insert 1.5ul 3ul 3ul 2ul
ddH2O 6.5ul 5ul 5ul 6ul
2x Quick ligase buffer 10ul 10ul 10ul 10ul
Quick ligase 1ul 1ul 1ul 1ul
Total 21ul 21ul 21ul 21ul
  1. Incubate at room temperature for 10 min.
  2. Take 10ul ligation system solution to do transformation.

9.3

Picking single colonies of bla, bleo, puro, zeo

Time: 18:00

Handler: Lu Shixin

Procedure:

  1. Pick single colonies, adding 6ml LB with corresponding antibiotics.
  2. Incubate overnight at 37℃, 220rpm.

9.4

Plasmid extraction of bla, bleo, puro, zeo

Time: 11:30

Handler: Lu Shixin

Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol

Result:

Concentration: bla②: 154.7 ng/ul 1.82; bleo①: 27.8 ng/ul 2.03; bleo②: 176.8 ng/ul 1.84; puro①: 20.9 ng/ul 2.06; puro②: 249.4 ng/ul 1.74; zeo①: 121.4 ng/ul 1.94; zeo②: 154.2 ng/ul 1.85.

Enzyme Digestion and Go Gel Electrophoresis Test of bla, bleo, puro, zeo

Time: 17:00

Handler: Lu Shixin

Procedure:

  1. Make enzyme cutting system solution.
bla② bleo① bleo② puro① puro② zeo① zeo②
DNA 2ul 8.8ul 2ul 8.8ul 1ul 2ul 2ul
Cutsmart buffer 1ul 1ul 1ul 1ul 1ul 1ul 1ul
EcoRI-HF 0.1ul 0.1ul 0.1ul 0.1ul 0.1ul 0.1ul 0.1ul
PstI-HF 0.1ul 0.1ul 0.1ul 0.1ul 0.1ul 0.1ul 0.1ul
ddH2O 6.8ul 0ul 6.8ul 0ul 7.8ul 6.8ul 6.8ul
Total 10ul 10ul 10ul 10ul 10ul 10ul 10ul
bla② bleo① bleo② puro① puro② zeo① zeo②
DNA 1ul 1ul 1ul 1ul 1ul 1ul 1ul
Cutsmart buffer 1ul 1ul 1ul 1ul 1ul 1ul 1ul
EcoRI-HF 0.1ul 0.1ul 0.1ul 0.1ul 0.1ul 0.1ul 0.1ul
ddH2O 7.9ul 7.9ul 7.9ul 7.9ul 7.9ul 7.9ul 7.9ul
Total 10ul 10ul 10ul 10ul 10ul 10ul 10ul
  1. Incubate 1h at 37℃.
  2. Make 1% agarose gel 50ml.
  3. Add 6x loading dye to the enzyme cutting system solution.
  4. Go gel electrophoresis.

Results:

530x364px

529x351px

9.12

Site–Directed Mutagenesis of bleo②

Time: 20:30

Handler: Tang Shiqiang

Procedure:

DNA 1ul
Mbleo1a 1.25ul
Mbleo1b 1.25ul
PrimeSTAR 25ul
ddH2O 21.5ul
Total 50ul
30 cycles Temperature Time
Initialization 98℃ 30s
Denaturation 98℃ 10s
Annealing 55℃ 5s
Extension 72℃ 13s
Final elongation 72℃ 2min

Cycle Pure of mbleo

Time: 22:20

Handler: Tang Shiqiang

Procedure: bio-tek OMEGA E.Z.N.A. Cycle Pure Kit Centrifugation Protocol

Result:

Concentration: mbleo: 116 ng/ul.

Enzyme Digestion for Gel Extraction of mbleo

Time: 23:30

Handler: Tang Shiqiang

Procedure:

  1. Make enzyme cutting system solution.
mbleo
DNA（>4ug） 44.5ul
Cutsmart buffer 5ul
DpnI 0.5ul
ddH2O 0ul
Total 50ul
  1. Incubate overnight at 37℃.

9.13

Transformation of mbleo

Time: 15:00

Handler: Tang Shiqiang

Procedure: IGEM Protocols

9.14

Picking single colonies of mbleo

Time: 10:30

Handler: Tang Shiqiang

Procedure:

  1. Pick single colonies, adding 6ml LB with corresponding antibiotics.
  2. Incubate about 12h at 37℃, 220rpm.

Plasmid extraction of mbleo

Time: 23:10

Handler: Tang Shiqiang

Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol

Result:

Concentration: mbleo①: 104.7 ng/ul 1.89; mbleo②: 94.7 ng/ul 2.03; mbleo③: 95.4 ng/ul 1.98; mbleo④: 96.4 ng/ul 2.01.

9.15

Enzyme Digestion and Go Gel Electrophoresis Test of mbleo

Time: 19:30

Handler: Lu Shixin

Procedure:

  1. Make enzyme cutting system solution.
bleo mbleo① mbleo② mbleo③ mbleo④
DNA 3ul 3ul 3ul 3ul 3ul
Cutsmart buffer 1ul 1ul 1ul 1ul 1ul
XbaI 0.1ul 0.1ul 0.1ul 0.1ul 0.1ul
PstI-HF 0.1ul 0.1ul 0.1ul 0.1ul 0.1ul
ddH2O 5.8ul 5.8ul 5.8ul 5.8ul 5.8ul
Total 10ul 10ul 10ul 10ul 10ul
bleo mbleo① mbleo② mbleo③ mbleo④
DNA 2ul 2ul 2ul 2ul 2ul
Cutsmart buffer 1ul 1ul 1ul 1ul 1ul
XbaI 0.1ul 0.1ul 0.1ul 0.1ul 0.1ul
ddH2O 6.9ul 6.9ul 6.9ul 6.9ul 6.9ul
Total 10ul 10ul 10ul 10ul 10ul
  1. Incubate 1h at 37℃.
  2. Make 1% agarose gel 50ml.
  3. Add 6x loading dye to the enzyme cutting system solution.
  4. Go gel electrophoresis.

Results:

553x412px

10.5

PCR of bla-2A, 2A-bleo, puro-2A

Time: 11:00

Handler: Tang Shiqiang

Procedure:

DNA 1ul
Primer1 1ul
Primer 2 1ul
PrimeSTAR 20ul
ddH2O 17ul
Total 40ul
30 cycles Temperature Time
Initialization 98℃ 30s
Denaturation 98℃ 10s
Annealing 55℃ 10s
Extension 72℃ 25s
Final elongation 72℃ 2min

Go Gel Electrophoresis and Gel Extraction of bla-2A, 2A-bleo, puro-2A PCR Product

Time: 19:00

Handler: Tang Shiqiang

Procedure: bio-tek OMEGA E.Z.N.A. Gel Extraction Kit – Spin Protocol

378x373px

Concentration: bla-2A: 31.1 ng/ul 3.19; 2A-bleo: 47.4 ng/ul 2.28; puro-2A: 73.0 ng/ul 2.32.

Enzyme Digestion for Gel Extraction of bla-2A, 2A-bleo, puro-2A

Time: 19:30

Handler: Tang Shiqiang

Procedure:

  1. Make enzyme cutting system solution.
bla-2A 2A-bleo puro-2A
DNA（>1ug） 44ul 44ul 44ul
Cutsmart buffer 5ul 5ul 5ul
EcoRI-HF 0.5ul 0.5ul 0.5ul
SpeI-HF 0.5ul 0.5ul 0.5ul
ddH2O 0ul 0ul 0ul
Total 50ul 50ul 50ul
  1. Incubate for 5h at 37℃.

10.6

Cycle Pure of bla-2A, 2A-bleo, puro-2A

Time: 1:00

Handler: Tang Shiqiang

Procedure: bio-tek OMEGA E.Z.N.A. Cycle Pure Kit Centrifugation Protocol

Results:

Concentration: bla-2A: 72.4 ng/ul 1.89; 2A-bleo: 74.4 ng/ul 1.88; puro-2A: 175.3 ng/ul 1.91.

Ligation of bla-2A, 2A-bleo, puro-2A to pSB1C3

Time: 1:30

Handler: Tang Shiqiang

Procedure:

  1. Make ligation system solution.
bla-2A 2A-bleo puro-2A
Vector(50ng) 1ul 1ul 1ul
Insert 0.7ul 0.7ul 0.3ul
ddH2O 3.3ul 3.3ul 3.7ul
2x Quick ligase buffer 5ul 5ul 5ul
Quick ligase 0.5ul 0.5ul 0.5ul
Total 10.5ul 10.5ul 10.5ul
  1. Incubate at room temperature for 10 min.
  2. Take 10ul ligation system solution to do transformation.

Picking single colonies of bla-2A, 2A-bleo, puro-2A

Time: 19:30

Handler: Tang Shiqiang

Procedure:

  1. Pick single colonies, adding 6ml LB with corresponding antibiotics.
  2. Incubate overnight at 37℃, 220rpm.

10.7

Plasmid extraction of bla-2A, 2A-bleo, puro-2A

Time: 11:00

Handler: Tang Shiqiang

Procedure: bio-tek OMEGA E.Z.N.A. Plasmid DNA Mini Kit I Spin Protocol

Result:

Concentration: bla-2A①: 266.9 ng/ul 1.94; bla-2A②: 249.3 ng/ul 1.95; 2A-bleo①: 261.2 ng/ul 1.95; 2A-bleo②: 224.5 ng/ul 1.96; puro-2A①: 223.6 ng/ul 1.99; puro-2A②: 170.2 ng/ul 1.98.

Enzyme Digestion and Go Gel Electrophoresis Test of bla-2A, 2A-bleo, puro-2A

Time: 13:30

Handler: Tang Shiqiang

Procedure:

  1. Make enzyme cutting system solution.
bla-2A① bla-2A② 2A-bleo① 2A-bleo② puro-2A① puro-2A②
DNA 3.6ul 3.9ul 1.3ul 1.6ul 3.5ul 4.5ul
Cutsmart buffer 1ul 1ul 1ul 1ul 1ul 1ul
EcoRI-HF 0.2ul 0.2ul \ \ 0.2ul 0.2ul
PvuI-HF 0.2ul 0.2ul \ \ \ \
StuI \ \ \ \ 0.2ul 0.2ul
ApaLI \ \ 0.3ul 0.3ul \ \
ddH2O 5ul 4.7ul 7.4ul 7.1ul 5.1ul 4.1ul
Total 10ul 10ul 10ul 10ul 10ul 10ul
  1. Incubate 1h at 37℃.
  2. Make 1% agarose gel 50ml.
  3. Add 6x loading dye to the enzyme cutting system solution.
  4. Go gel electrophoresis.

Results:

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