8 Chromoprotein

Plasmid Construction

ALL ABBREVIATIONS USED:

Name	Number	Index	Position	Backbone	Length
Strong promoter	①	BBa_J23100	4-17-D	BBa_J61002	35
Weak promoter	②	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

2. Incubate at 37℃ for 30 min.

3. Make 1% agarose gel 30ml.

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

5. Go gel electrophoresis.

Results:

figures show below:

7.28

Competent Cell Test Colonies Counting

Time: 13:00

Handler: Lu Shixin

Result:

{{SUSTech_Image_Center_10 | filename=SUSTech_Shenzhen-372E5718-44FE-463A-86A8-57C609A8F345.png | caption= ]] 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：

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:

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:

‘’‘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:

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

2. 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:

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:

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:

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:

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:

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:

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:

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

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:

GECO

9.23

PCR of GECO

Time: 15:00

Handler: Tang Shiqiang

Procedure:

DNA	1ul
Forward	1ul
Reverse	1ul
PrimeSTAR	20ul
ddH2O	17ul
Total	40ul

30 cycles	Temperature	Time
Initialization	98℃	30s
Denaturation	98℃	5s
Annealing	55℃	5s
Extension	72℃	30s
Final elongation	72℃	2min

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

Time: 19:30

Handler: Tang Shiqiang

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

Result:

Concentration: 84.1 ng/ul 1.77.

Enzyme Digestion for Gel Extraction of GECO

Time: 20:00

Handler: Tang Shiqiang

Procedure:

1. Make enzyme cutting system solution.

	GECO
DNA（>3ug）	44ul
Cutsmart buffer	5ul
EcoRI-HF	0.5ul
SpeI-HF	0.5ul
ddH2O	0ul
Total	50ul

1. Incubate for 5h at 37℃.

9.24

Cycle Pure of GECO

Time: 1:00

Handler: Tang Shiqiang

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

Results:

Concentration: GECO: 133.4 ng/ul 1.85.

Ligation of GECO to pSB1C3

Time: 1:30

Handler: Tang Shiqiang

Procedure:

1. Make ligation system solution.

	GECO
Vector(50ng)	0.4ul
Insert	0.4ul
ddH2O	4.2ul
2x Quick ligase buffer	5ul
Quick ligase	0.5ul
Total	10.5ul

1. Incubate at room temperature for 10 min.
2. Take 10ul ligation system solution to do transformation.

Picking single colonies of GECO

Time: 19:30

Handler: Tang Shiqiang

Procedure:

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

9.25

Plasmid extraction of GECO

Time: 10:20

Handler: Tang Shiqiang

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

Result:

Concentration: GECO①: 352.8 ng/ul 1.81; GECO②: 360.7 ng/ul 1.73.

Enzyme Digestion and Go Gel Electrophoresis Test of GECO

Time: 14:30

Handler: Tang Shiqiang

Procedure:

1. Make enzyme cutting system solution.

	GECO①	GECO②
DNA	2ul	2ul
Cutsmart buffer	1ul	1ul
SbfI-HF	0.5ul	0.5ul
BspDI	0.5ul	0.5ul
ddH2O	6ul	6ul
Total	10ul	10ul

1. Incubate 1h at 37℃.
2. Make 1% agarose gel 30ml.
3. Add 6x loading dye to the enzyme cutting system solution.
4. Go gel electrophoresis.

Results:

Site–Directed Mutagenesis of GECO①

Time: 16:20

Handler: Tang Shiqiang

Procedure:

DNA	1ul
Mpst1a	1ul
Mpst1b	1ul
PrimeSTAR	20ul
ddH2O	17ul
Total	40ul

30 cycles	Temperature	Time
Initialization	98℃	30s
Denaturation	98℃	10s
Annealing	55℃	5s
Extension	72℃	90s
Final elongation	72℃	2min

Cycle Pure of M1GECO

Time: 20:00

Handler: Tang Shiqiang

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

Result:

Concentration: M1GECO: 118.8 ng/ul 1.83.

Enzyme Digestion for Gel Extraction of M1GECO

Time: 20:30

Handler: Tang Shiqiang

Procedure:

1. Make enzyme cutting system solution.

	M1GECO	GECO control
DNA（>4ug）	44.5ul	5ul
Cutsmart buffer	5ul	5ul
DpnI	0.5ul	0.5ul
ddH2O	0ul	43.5ul
Total	50ul	50ul

1. Incubate for 2h at 37℃.

9.26

Transformation of M1GECO

Time: 0:40

Handler: Tang Shiqiang

Procedure: IGEM Protocols

Picking single colonies of M1GECO

Time: 19:00

Handler: Tang Shiqiang

Procedure:

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

9.27

Plasmid extraction of M1GECO

Time: 12:00

Handler: Lu Shixin

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

Result:

Concentration: M1GECO①: 262.7 ng/ul 1.71; M1GECO②: 277.1 ng/ul 1.60.

Site–Directed Mutagenesis of M1GECO

Time: 13:00

Handler: Tang Shiqiang

Procedure:

DNA	1ul
Mpst2a	1ul
Mpst2b	1ul
PrimeSTAR	20ul
ddH2O	17ul
Total	40ul

30 cycles	Temperature	Time
Initialization	98℃	30s
Denaturation	98℃	10s
Annealing	55℃	5s
Extension	72℃	90s
Final elongation	72℃	2min

Cycle Pure of M2GECO

Time: 17:00

Handler: Lu Shixin

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

Result:

Concentration: M2GECO: 100.0 ng/ul 1.83.

Enzyme Digestion for Gel Extraction of M2GECO

Time: 17:30

Handler: Lu Shixin

Procedure:

1. Make enzyme cutting system solution.

	M2GECO	M1GECO control
DNA（>4ug）	44.5ul	5ul
Cutsmart buffer	5ul	5ul
DpnI	0.5ul	0.5ul
ddH2O	0ul	43.5ul
Total	50ul	50ul

1. Incubate for 3h at 37℃.

Transformation of M2GECO

Time: 20:30

Handler: Lu Shixin

Procedure: IGEM Protocols

9.28

Picking single colonies of M2GECO

Time: 10:00

Handler: Tang Shiqiang

Procedure:

1. Pick single colonies, adding 6ml LB with corresponding antibiotics.
2. Incubate for 13h at 37℃, 220rpm.

Plasmid extraction of M2GECO

Time: 23:00

Handler: Lu Shixin

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

Result:

Concentration: M2GECO①: 175.1 ng/ul 1.70; M2GECO②: 117.5 ng/ul 1.86; M2GECO③: 102 ng/ul 1.90.

9.29

=Enzyme Digestion and Go Gel Electrophoresis Test of M2GECO

Time: 0:00

Handler: Lu Shixin

Procedure:

1. Make enzyme cutting system solution.

	M2GECO①	M2GECO②	M2GECO③
DNA	1ul	1ul	1ul
Cutsmart buffer	1ul	1ul	1ul
PstI-HF	0.1ul	0.1ul	0.1ul
ddH2O	7.9ul	7.9ul	7.9ul
Total	10ul	10ul	10ul

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:

TetOn

9.26

PCR of GECO

Time: 20:00

Handler: Tang Shiqiang

Procedure:

DNA	1ul
Forward	1ul
Reverse	1ul
PrimeSTAR	20ul
ddH2O	17ul
Total	40ul

30 cycles	Temperature	Time
Initialization	98℃	30s
Denaturation	98℃	10s
Annealing	55℃	5s
Extension	72℃	40s
Final elongation	72℃	2min

Go Gel Electrophoresis and Gel Extraction of TetOn PCR product

Time: 21:30

Handler: Lu Shixin

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

Result:

Concentration: TetOn: 86.7 ng/ul 1.66.

Enzyme Digestion for Gel Extraction of TetOn

Time: 22:30

Handler: Lu Shixin

Procedure:

1. Make enzyme cutting system solution.

	TetOn
DNA（>3ug）	44ul
Cutsmart buffer	5ul
EcoRI-HF	0.5ul
SpeI-HF	0.5ul
ddH2O	0ul
Total	50ul

1. Incubate overnight at 37℃.

9.27

Cycle Pure of TetOn

Time: 12:00

Handler: Lu Shixin

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

Results:

Concentration: TetOn: 45.4 ng/ul 1.80.

Ligation of TetOn to pSB1C3

Time: 15:00

Handler: Tang Shiqiang

Procedure:

1. Make ligation system solution.

	TetOn
Vector(50ng)	0.4ul
Insert	1.6ul
ddH2O	3ul
2x Quick ligase buffer	5ul
Quick ligase	0.5ul
Total	10.5ul

1. Incubate at room temperature for 10 min.
2. Take 10ul ligation system solution to do transformation.
3. Put the plate at 4℃ after 16h.

9.29

Picking single colonies of TetOn

Time: 20:00

Handler: Tang Shiqiang

Procedure:

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

9.30

Plasmid extraction of TetOn

Time: 10:30

Handler: Tang Shiqiang

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

Result:

Concentration: TetOn①: 246.6 ng/ul 1.79; TetOn②: 314.1 ng/ul 1.86; TetOn③: 106.3 ng/ul 1.86; TetOn④: 298.8 ng/ul 1.86.

Enzyme Digestion and Go Gel Electrophoresis Test of TetOn

Time: 14:30

Handler: Tang Shiqiang

Procedure:

1. Make enzyme cutting system solution.

	TetOn①	TetOn②	TetOn③	TetOn④
DNA	2.5ul	2.5ul	6ul	2.5ul
Cutsmart buffer	1ul	1ul	1ul	1ul
EcoRI-HF	0.2ul	0.2ul	0.2ul	0.2ul
SacII	0.2ul	0.2ul	0.2ul	0.2ul
ddH2O	6.1ul	6.1ul	2.6ul	6.1ul
Total	10ul	10ul	10ul	10ul

1. Incubate 1h at 37℃.
2. Make 1% agarose gel 30ml.
3. Add 6x loading dye to the enzyme cutting system solution.
4. Go gel electrophoresis.

Results: