Notebooks

7.27

V.natriegens activation

XIRAN HU

Freeze-dried V.natriegens ATCC14048 ordered from CCIC was activated in LB+v2 salts overnight.

7.28

XIRAN HU

Glycerol stock

JINXUAN LU, JINPENG LI

Overnight culture of V.natriegens was stored as glycerol stock for later use.

Growth curve measurement

Three different mediums were inoculated with overnight V.natriegens culture to measure the growth curve. The result was as follows:

8.3

Stab culture activation

XIRAN HU

Top 10 strains carrying plasmids ordered from Azenta are activated from stab culture into liquid culture(LB broth with appropriate antibiotics).

8.4

ZIWEN XU

Plasmid extraction

XIRAN HU

Plasmids pGFP, pdCas9-sg1 and pPilin were extracted from Top 10 liquid culture. Concentration and quality of the plasmids were measured with Nanodrop.

	pGFP	pdCas9-sg1	pPilin
concentration(ng/μL)	352.9	38.5	371.8
OD260/280	1.89	1.77	1.90
OD260/230	2.18	2.31	1.15

pdCas9-sg1 can not be used for transformation.

Glycerol stock

XIRAN HU

3 Top 10 strains were stored as glycerol stock for later use.

Chemical competent cell preparation

YUCHENG ZHANG

Prepare chemical competent V.natriegens for transformation.The chemical competent cells were stored at -80℃ until use.

8.5

Transformation of chemical competent cells

ZIWEN XU

The transformation was performed on home-made chemical competent V.natriegens and comercial DH5α with the following parameters

number	Cell type	plasmid name	Plasmid concentration(μg/μL)	Plasmid quantity(μL)
1	DH5α	pPilin	371.8	1
2	DH5α	pPilin	37.18	2
3	V.natriegens	pPilin	371.8	1
4	V.natriegens	pPilin	37.18	2
5	DH5α	pGFP	352.9	1
6	DH5α	pGFP	35.29	2
7	V.natriegens	pGFP	352.9	1
8	V.natriegens	pGFP	35.29	2

8.6

XIRAN HU

Colony PCR

The result of the overnight Agar plate cultures was as follows.There were several colonies on the plates corresponding to DH5α transformations but only one colony on transformation 7.

Several colonies were selected to conduct colony PCR with appropriate primers.The result is as follows:

All positive strains were stored as glycerol stock. DH5α cells transformed with pPilin were designated as EP1, EP2, EP3 and DH5α cells transformed with pGFP were designated as EG1, EG2, EG3

EP2 overnight culture

XIRAN HU

Strain EP2 was inoculated in 2mL of LB broth culture with 100μg/mL of Ampicilin for pilin induction.

Transformation of chemical competent cells

XIRAN HU

The transformation was performed on home-made chemical competent V.natriegens with the following parameters

number	Cell type	plasmid name	Plasmid concentration(μg/μL)	Plasmid quantity(μL)
1	V.natriegens	pGFP	352.9	1
2	V.natriegens	pGFP	35.29	2

8.7

Colony PCR

YUCHENG ZHANG

There were 2 colonies on the plate corresponding to transformation 1. Colony PCR was conducted on these 2 colonies with appropriate primers. But no band was seen.

EP2 extended culture and pilin induction

YUCHENG ZHANG

2×30 mL of LB broth with 100μg/mL of Ampicilin were inoculated with overnight EP2 liquid culture. Arabinose solution, the inducer, was added to one tube when OD600 of the liquid culture in it reached 0.5. And then the culture continued for 5h before harvest.

Protein extraction from EP2

ZIWEN XU

Soluable proteins were extracted and collected from EP2 after the induction.

Pilus harvest

XIRAN HU

Pilus were harvested according to the protocol from EP2 culture.

BCA test of soluable proteins extracted from EP2

XIRAN HU

BCA test of soluable proteins extracted from EP2

8.9

ZIWEN XU

Whole cell lysates WB for Pilin identification

Overnight culture of EP2 was inoculated to the following culture mediums, 1.5mL for each condition:

Culturing time\induction concentration	0	10mM	20mM
10h	1	2	3
24h	4	5	6

The induction of pilin expression was performed when the OD600 reached 0.5 by arabinose solution.

After stained with coomassie brilliant blue, no significant difference was seen between groups. The following steps were performed according to the protocols. No band was seen.

8.12

XIRAN HU

Glycerol stocks of the top 10 strains carrying plasmids ordered from Azenta were inoculated into liquid culture (LB broth with appropriate antibiotics). A glycerol stock of wild type Vibrio natriegens was inoculated into LB+v2 salt liquid medium for tomorrow's electrocompetent cells preparation.

8.13

Plasmid extraction

ZIWEN XU

Plasmids pGFP, pdCas9-sg1 and pPilin were extracted from Top 10 liquid culture. Concentrations and qualities of the plasmids were measured with Nanodrop.

	pGFP	pdCas9-sg1	pPilin
concentration(ng/μL)	220.5	159.7	180.3
OD260/280	1.82	1.88	1.88
OD260/230	1.95	2.01	2.14

Ectrocompetent cells preparation(protocol 1)

YUCHENG ZHANG

Electrocompetent Vibrio natriegens were prepared as described in the protocol.

Electroporation

XIRAN HU

Electroporation was performed according to the protocol with the following parameters:

number	voltage(v)	time(ms)
1	700	1
2	700	2
3	700	3
4	700	4

8.14

YUCHENG ZHANG

Electroporation result

There was no colony on the plates.

Electrocompetent cells preparation(protocol 1)

Electrocompetent Vibrio natriegens were prepared as described in the protocol.

8.15

XIRAN HU

Electroporation

Electroporation was performed according to the protocol with the following parameters:

number	voltage(v)	time(ms)
1	700	1
2	700	2
3	800	1
4	800	1.7
5	820	1.8
6	900	1

Whole cell lysates WB for pilin identification

XIRAN HU

Overnight culture of EP2 was inoculated into the following mediums:

Number	Inducer concentration	Culturing time after induction
1	0mM	5h
2	10mM	5h
3	20mM	5h
4	0mM	15h
5	10mM	15h
6	20mM	15h

After transfer, the PVDF membrane was stained with Ponceau S. No band was seen, perhaps due to the unstable current of the electrophoresis system.

8.17

XIRAN HU

Electroporation result

There was no colony on the plates.

electroporation

Electroporation was performed according to the protocol with the following parameters:

number	voltage(v)	time(ms)
1	700	2.2
2	790	2.2
3	900	2.2
4	800	2.3
5	740	2.2
6	840	2.2

Transformation of chemical competent BL21(DE3) cells

ZIWEN XU

Comercial chemical competent BL21(DE3) cells were transformed with 3μL(684 ng) of pPilin according to the protocol.

8.18

Colony PCR

YUCHENG ZHANG

There were several colonies on the plate of BL21 transformation. Colony PCR was conducted on 4 of these colonies with appropriate primers. The result was as follows.

Strain corresponding to band 1 was designated as BP1.

BP2 extended culture and pilin induction

XIRAN HU

2×30 mL of LB broth with 100μg/mL of Ampicilin were inoculated with overnight EP2 liquid culture. Arabinose solution, the inducer, was added to one tube when OD600 of the liquid culture in it reached 0.5. And then the culture continued for 5h and 15h before harvest.

8.19

XIRAN HU

Whole cell lysates SDS-PAGE for pilin identification

Samples for SDS-PAGE were prepared from the following mediums:

number	Inducer concentration	Culturing time after induction
1	0mM	5h
2	20mM	5h
3	0mM	15h
4	20mM	15h

No significant difference in the molecular weight of pilin was seen between the lanes.

Electrocompetent cell preparation (protocol 2)

YUCHENG ZHANG

Electrocompetent cells were prepared according to the protocol.

Electroporation

XIRAN HU

Electroporation was performed according to the protocol with the following parameters:

Plasmid	Plasmid volume(μL)	voltage(v)	time(ms)
pPililn	5	700	2.2
pPilin	5	790	2.2
pGFP	5	900	2.2
pGFP	5	800	2.3
pdCas9-sg1	5	740	2.2
pdCas9-sg1	5	840	2.2

8.20

YUCHENG ZHANG

Colony PCR

There were several colonies on the plates. Colony PCR was conducted on 11 of these colonies with appropriate primers. The result was as follows.

Strains tested positive were designated as V.CR1, V.CR2, V.CR3, V.CR4, V.CR5, V.PL V.GF3 and V.GF4.

8.21

V. CR1 extended culture and dCas9 induction

ZIWEN XU

Overnight V. CR1 culture was inoculated into the following mediums.Inducer was added when the OD600 reached 0.5.

number	Inducer concentration(mg/mL)	Culturing time after induction
1	0	5h
2	0.5	5h
3	1	5h
4	0	15h
5	0.5	15h
6	1	15h

8.22

ZIWEN XU

Whole cell lysates SDS-PAGE for dCas9 identification

1 mL of each V.CR1 culture was centrfuged and the pellets were resuspended with 35 μL of ddH₂O.

10 μL of 5×loading buffer was added to each of the tubes and the tubes were incubated at 95℃ for 5 min. 10 μL of prepared samples were loaded.

There was no significant difference in the molecular weight of dCas9 between lanes.

8.24

Green fluorescence observation

YUCHENG ZHANG

Overnight V.GF4 culture and Vibrio natriegens 14048 culture were enlarged for 2 hours.

The fluorescence was observed with fluorescence microscope. There was no significant difference between strain 14048 and V.GF4.

Pilin induction in V.PL

XIRAN HU

Overnight V.PL culture was inoculated into the following mediums at a dilution ratio of 1:100. Induction was performed 2h after the inoculation.

number	inducer(arabinose) concentration	Culturing time after induction	Medium volume	temperature
1	0mM	11h	30mL	37℃
2	0mM	11h	30m	37℃
3	20mM	11h	30mL	37℃
4	20mM	11h	30mL	37℃

8.25

XIRAN HU

Pilus harvest

Culture number 1 and 2 were consolidated while number 3 and 4 were consolidated. Pilus were harvested according to the protocol. The BCA test result of the two acquired samples was as follows.

Sample1 was from uninduced culture while sample2 was from induced culture. The final concentrations of the two samples were 0.39μg/μL and 0.28μg/μL, respectively.

1.5mL of sample1 and sample 2 were centrifuged to prepare whole cell lysate samples designated as sample1-1 and sample2-1

Pilin WB

XIRAN HU

WB of pilin was performed on sample1, sample1-1, sample2 and sample2-1 according to the protocol.

8.26

WB result

XIRAN HU

There was no band on the membrane. The secondary antibody was not adequately washed off and small protein bands were too obscure to be observed. Tris-tricine SDS-PAGE should be used for pilin WB.

Extended culture of V.CR1 and dCas9 induction

ZIWEN XU

Overnight V.CR1 culture was inoculated into the following mediums. Inducer was added when the OD600 reached 0.5.

number	Inducer concentration(mg/mL)	Culturing time after induction
1	0	6h
2	0.5	6h

Whole cell lysates SDS-PAGE for dCas9 identification

ZIWEN XU

1mL of each culture above was centrifuged and the pellets were resusppended with 35μL of ddH₂O. 5uL of each of the prepared samples were loaded for SDS-PAGE. The result is as follows. The band marked with * was darker on the induced lane and the molecular weight of dCas9(about 150kDa) is between 180kDa and 130kDa.

8.27

Whole cell lysates SDS-PAGE for sfGFP identification

YUCHENG ZHANG

1 mL of overnight V.GF4 culture and 14048 culture were centrifuged and the pellets were resuppended with ddH₂O. 5 μL of 5×loading buffer was added to the tubes which were then incubated at 95℃ for 5mins. 5 μL of each of the prepared samples was loaded for SDS-PAGE.

There were too many bands to distinguish the differences.

Electrocompetent cell preparation (protocol 2)

YUCHENG ZHANG

Overnight V.PL and V.CR1 culture were used for electrocompetent cell preparation according to the protocol.

Electroporation

XIRAN HU

Electroporation was performed according to the following parameters.

number	Competent cell	plasmid	Plasmid volume(μL)	time(ms)	voltage(v)	antibiotics
1	V.CR1	pPilin	5	4	790	Ampicilin
2	V.CR1	pPilin	5	4	790	Ampicilin
3	V.PL	pdCas9-sg1	5	3.8	790	Kanamycin
4	V.PL	pdCas9-sg1	5	4	790	Kanamycin

8.28

V. CR1 extended culture and dCas9 induction

ZIWEN XU

Overnight V. CR1 and ATCC14048 culture was inoculated into the following mediums. Inducer was added when the OD600 reached 0.5.

number	cell	Inducer concentration(mg/mL)	Culturing time after induction
1	WT	0	5h
2	V.CR1	0	5h
3	V.CR1	0.5	5h
4	V.CR1	0.5	10h

SDS-PAGE of dCas9

ZIWEN XU

Soluable proteins were extracted from 1.5 mL of the cultures above. The result of SDS-PAGE was as follows:

There was no significant difference between the 4 lanes.

SDS-PAGE of sfGFP

YUCHENG ZHANG

1 mL of overnight V.GF4 and ATCC14048 culture were centrifuged and then resuspended with 35 μL of ddH₂O. 1.5 mL of overnight V.GF4 and ATCC14048 culture were used for soluable protein extraction. The result of SDS-PAGE was as follows.

There was a significant darker band on the lane of V.GF4 soluable proteins(marked with).The molecular weight of the modified sfGFP is about 31kD, between 25kD and 35kD. The experiment would be repeated 2 times to ensure the result.

9.8

pGFP modification

YUCHENG ZHANG

Reversed PCR

Component	Volume
2 × Phanta Flash Master Mix	25μL
ddH2O	20μL
CutF+R(5μM)	4μL
pGFP	1μL
annealing temperature	59℃
elongation time	20sec

Gel extraction

The linearized pGFPcut was purified from PCR mix.

Gibson assembly

Component	Volume
Linearized pGFP	1 μL(333ng)
2×CE mix	5μL
ddH2O	4μL

The recombinant product (desingated as pGFPcut) was transformed into electrocompetent V.natriegens cells.

SDS-PAGE of sfGFP

XIRAN HU

1 mL of overnight V.GF4 and ATCC14048 culture were centrifuged and then resuspended with 35 μL ddH₂O. 1.5mL overnight V.GF4 and ATCC14048 culture were used for soluable protein extraction. The result of SDS-PAGE was as follows.

There was a significantly darker band in the lane of V.GF4 soluable proteins (marked with'*').The molecular weight of the modified sfGFP is about 31kD, between 25kD and 35kD. The experiment will be repeated one more time to confirm the result.

Electroporation

XIRAN HU

Electroporation was performed according to the following parameters.

number	Competent cell	plasmid	Plasmid volume(μL)	time(ms)	voltage(v)	antibiotics
1	V.CR1	pPilin	5	3.7	790	Amp(40μg/mL)
2	V.PL	pdCas9-sg1	5	4	790	Kan(50μg/mL)

Pilin induction in V.PL

XIRAN HU

Overnight V.PL culture was inoculated into the following mediums at a dilution ratio of 1:100. Induction was performed 2h after the inoculation.

number	Cell type	inducer(arabinose) concentration	Culturing time after induction	Medium volume	temperature
1	WT	20mM	16h	30mL	37℃
2	V.PL	20mM	16h	30m	37℃

9.9

YUCHENG ZHANG

Colony PCR

There were several colonies on the plate supplemented with Kanamycin. 2 colonies were chosen to conduct colony PCR with the following parameters：

pPilin verification
Component	Volume
2 × Taq Master Mix	25μL
ddH2O	20μL
PLR+PLF(5μM)	4μL
colony	1

pdCas9-sg1 verification
Component	Volume
2 × Taq Master Mix	25μL
ddH2O	20μL
CRR+CRF(5μM)	4μL
colony	1

Bands were only seen on the lanes corresponding to pdCas9-sg1 verification. The electroporation was not successful.

SDS-PAGE of Pilin

XIRAN HU

1.5 mL of each of the 4 cultures were used for soluable protein extraction. 10μL of prepared sample was loaded. The result of SDS-PAGE was as follows. The issue may lie in the gel or loading buffer.

9.10

SDS-PAGE of sfGFP

YUCHENG ZHANG

1 mL of overnight V.GF4 and ATCC14048 culture were centrifugedand then resuspended with 35 μL ddH₂O. 1.5mL of overnight V.GF4 and ATCC14048 culture were used for soluable protein extraction. The result of SDS-PAGE was as follows. a stands for all proteins and s stands for extracted soluable proteins.

There was a significant darker band on the lane of V.GF4 soluable proteins (marked with'*' ).The molecular weight of the modified sfGFP is about 31kD, between 25kD and 35kD.

Gibson assembly

ZIWEN XU

The linearized pGFP solution was diluted to 67.26 μg/mL.

Component	Volume
Linearized pGFP(diluted)	4 μL
2×CE mix	5 μL
ddH2O	1 μL

Chemical and electrocompetent cell prepareation (protocol 2)

YUCHENG ZHANG

Electroporation competent V.PL and V.CR1 were prepared according to the protocols.

Electroporation

Electroporation was performed according to the protocol with the following parameters.

Cell type	plasmid	Plasmid volume(μL)	voltage(v)	time(ms)
V.CR1	pPilin	5	790	4
V.CR1	pGFPcut	5	790	3.9
V.PL	pdCas9-sg1	5	790	4

Transformation of chemical competent DH5α

ZIWEN XU

2 μL of modified pET-24b(+) was added to 50 μL of chemical competent DH5α for transformation.

Pilin induction in V.PL

XIRAN HU

Overnight V.PL culture was inoculated into the following mediums at a dilution ratio of 1:100. Induction was performed 2h after the inoculation.

number	Cell type	inducer(arabinose) concentration	Culturing time after induction	Medium volume	temperature
1	WT	20mM	20h	2×30mL	16℃
2	V.PL	20mM	20h	2×30mL	16℃

9.11

XIRAN HU

Modified pET-24(+) carrying strain storage.

There was no colony on the plates of electroporation, maybe due to the overly used electroporation cuvettes. There were many colonies on the plate of DH5α transformation and 5 colonies were chosen to be strored.

Pilus harvest and SDS-PAGE of pilin

XIRAN HU

Pilus were harvested according to the protocol. The BCA test result of the harvested pilus solution was as follows.

According to the result, the concentration of sample1 (induced V.PL) was 0.46μg/μL and the concentration of sample2(WT) was 1.04μg/μL.

Electroporation

YICHEN LIU

Electroporation was performed according to the protocol with the following parameters.

Cell type	plasmid	Plasmid volume(μL)	voltage(v)	time(ms)
V.CR1	pPilin	5	790	4
V.PL	pdCas9-sg1	5	790	4

9.12

Colony PCR

XIRAN HU

5 colonies on the plates were chosen for colony PCR with the following parameters

pPilin verification
Component	Volume
2 × Taq Master Mix	25μL
ddH2O	20μL
PLR+PLF(5μM)	4μL
colony	1

pdCas9-sg1 verification
Component	Volume
2 × Taq Master Mix	25μL
ddH2O	20μL
CRR+CRF(5μM)	4μL
colony	1

The result was as follows. Strains that had bands in both PCR tests were stored as glycerol stock and designated as V.PC1, V.PC3, V.PC5, V.PC6 and V.PC7, respectively.

WB of harvested pilin

XIRAN HU

Step	Details
blocking	5% silk milk dissolved in TBST, 1.5h at RT
Primary antibody	1:4000, overnight at 4℃
Secondary antibody	1:5000, 1h at RT

9.13

WB of harvested pilin

The result of the WB was as follows. Marker bands of low molecular weight could not be seen, maybe because the transfer buffer was not supplemented with 20% of methanol.

Plasmid exraction

XIRAN HU

Plasmids were extracted from overnight culture of V.PC1, V.PC3, V.PC5, V.PC6, V.PC7 for sequencing.

Electroporation

YICHEN LIU

number	Cell type	plasmid	Plasmid volume	voltage	time
1	V.CR1	pGFPcut	5 μL	790v	4ms
2	ATCC14048	Control plasmid	5 μL	790v	4ms

V.CR1 extended culture and dCas9 induction

ZIWEN XU

Overnight V.CR1 culture was inoculated into the following mediums. Inducer was added when the OD600 reached 0.5.

number	Cell type	Inducer concentration(mg/mL)	Culturing time after induction	temperature
1	WT	0	20h	20 ℃
2	V.CR1	0.5	20h	20℃

9.15

pGFP modification

YUCHENG ZHANG

Reversed PCR
Component	Volume/Details
2 × Phanta Flash Master Mix	25μL
ddH2O	20 μL
modF1+R1(5μM)	4 μL
pGFP	1 μL
annealing temperature	53℃
elongation time	21sec

Gel extraction:Linearized pGFPmod1 was purified from PCR mix.

Gibson assembly
Component	Volume/Details
Linearized pGFP	2 μL(256ng)
2×CE mix	5 μL
ddH2O	3 μL

The recombinant product (desingated as pGFPmod1) was transformed into chemical competent DH5α cells according to the protocol.

Pilin induction in V.PC6

XIRAN HU

Overnight V.PC6 culture was inoculated into the following mediums at a dilution ratio of 1:100. Induction was performed 2h after inoculation.

Parameter	Details
Cell type	V.PC6
inducer(arabinose) concentration	20mM
Culturing time after induction	24h
Medium volume	2×200 mL
temperature	20℃

SDS-PAGE of dCas9

ZIWEN XU

1 mL of induced V.CR1 and ATCC14048 culture were centrifuged and then resuspended with 35 μL of ddH₂O. 1.5mL of overnight V.GF4 and ATCC14048 culture were used for soluable protein extraction. The result of SDS-PAGE was as follows. There was no significant difference between the two lanes.

9.16

Pilus harvest

XIRAN HU

Pilus were harvested according to the protocol. The BCA test result of the harvested pilus solutions was as follows. Concentration was 4.92μg/μL of sample1 , 4.62μg/μL of sample2, and 0.88μg/μL of sample3.

Chemical competent DH5α preparation

YUCHENG ZHANG

Chemical competent DH5α was prepared according to the protocols.

DH5α transformation

ZIWEN XU

Recombinant pGFPmod1 was transferomed into chemical competent DH5α cells accroding to the protocol.

9.17

XIRAN HU

WB of pilus

10 μL 2×tricine loading buffer was added to 40 μL of sample1 and sample2 respectively. Then the tubes were incubated at 95℃ for 5mins. 20.3μL of prepared sample1 and 21.8μL of prepared sample2 were loaded for SDS-PAGE. The primary antibody was diluted at 1:500.

Colony PCR

YUCHENG ZHANG

7 colonies on the plates were chosen for colony PCR with the following parameters

pPilin verification
Component	Volume/Details
2 × Taq Master Mix	25μL
ddH2O	20μL
GFR+GFF(5μM)	4μL
colony	1

The result was as follows.All colonies tested positive. Strains corresponding to band 1 and band 2 were chosen to conduct the following experiments and were designated as V.mod1-1 and V.mod1-2.

Pilin induction in V.PC6

XIRAN HU

Overnight V.PL culture was inoculated into the following mediums at a dilution ratio of 1:100. Induction was performed 2h after inoculation.

Cell type	V.PC6	ATCC14048
inducer(arabinose) concentration	20mM	20mM
Culturing time after induction	24h	24h
Medium volume	100 mL	100mL
temperature	20℃	20℃

9.18

WB of pilus

XIRAN HU

The secondary antibody was diluted at 1:5000. No band was seen on the membrane.

Gibson assembly and DH5α transformation

YICHENG ZHANG, ZIWEN XU

gibson assembly

Component	Volume/Details
Linearized pGFP	2 μL(256ng)
2×CE mix	5 μL
ddH2O	3 μL

The recombinant product (desingated as pGFPcut) was transformed into chemical competent DH5α cells according to the protocol.

9.19

Plasmid extraction

ZIWEN XU

Plasmids were extracted from overnight culture of V.mod1-1 and V.mod1-2.

Pilus harvest and SDS-PAGE of pilin

Pilus were harvested according to the protocol. The BCA test result was as follows. Sample 1 was harvested from V.PC6 culture and sample 2 was harvested from ATCC14048 culture.

Colony PCR

YUCHENG ZHANG

One colony on the plates was chosen for colony PCR with the following parameters.

pPilin verification
Component	Volume/Details
2 × Taq Master Mix	25μL
ddH2O	20μL
GFR+GFF(5μM)	4μL
colony	1

The result was as follows.The colony tested positive and was designated as V.cut.

pGFP modification

YUCHENG ZHANG

Reversed PCR

PCR Parameters
Component	Volume/Details
2 × Phanta Flash Master Mix	25μL
ddH2O	20 μL
modF2+R2(5μM)	4 μL
pGFPmod1	1 μL
annealing temperature	56℃
elongation time	21sec

Gel extraction

Linearized pGFPmod2 was purified from PCR mix.

Gibson assembly

Component	Volume/Details
Linearized pGFPmod1	2 μL(256ng)
2×CE mix	5 μL
ddH2O	3 μL

The recombinant product (desingated as pGFPmod2) was transformed into chemical competent DH5α cells according to the protocol.

DH5α transformation

ZIWEN XU

Recombinant pGFPmod2 was transferomed into chemical competent DH5α cells accroding to the protocol.

9.20

YUCHENG ZHANG

Chemical competent DH5α preparation

YUCHENG ZHANG

Chemical competent DH5α was prepared according to the protocols.

Plamisd extraction

XIRAN HU

Plamis was extracted from overnight culture of V.cut

DH5α transformation

XIRAN HU

Recombinant pGFPmod2 was transferomed into chemical competent DH5α cells accroding to the protocol.

9.21

Colony PCR

YUCHENG ZHANG

Five colonies on the plates was chosen for colony PCR with the following parameters.

pPilin verification
Component	Volume/Details
2 × Taq Master Mix	25μL
ddH2O	20μL
GFR+GFF(5μM)	4μL
colony	1

The result was as follows.One of the colonies tested positive was chosen for the following experiments.

Spread plate

XIRAN HU

Overnight V.PC1 culture was spread out on twenty agar plates supplement with kanacymin, ampicillin, 0.5% glycerol and 2% arabinose. 300μL of culture was spread for each plate.The plates were cultured at 37℃ for 24 h before pilus harvest.

Electroporation

XIRAN HU

Cell type	ATCC14048
plasmid	pGFPcut
Plasmid volume	5 μL
voltage	790 v
time	4 ms

9.22

Electroporation

YUCHENG ZHANG

no.	Cell type	plasmid	Plasmid volume	voltage	time
1	V.CR1	pGFPcut	5 μL	790 v	4 ms
2	V.CR1	pGFPmod2	5 μL	790 v	4 ms

Pilus harvest

XIRAN HU

The cells were harvested from the plates with 1 mL of LB+v2 salts medium for each plate.The following procedures were the same as the previous harvest protocol.

The concentration of harvested pilus solution was tested using the previous standard curve.The concentration of original pilus solution was about 4.8μg/μL

9.23

Electroporation

YICHEN LIU

no.	Cell type	plasmid	Plasmid volume	voltage	time
1	V.CR1	pGFPcut	5 μL	790 v	4 ms
2	V.CR1	pGFPmod2	5 μL	790 v	4 ms

electrocompetent cell prepareation (protocol 2)

YUCHENG ZHANG

Electroporation competent V.CR1 were prepared according to the protocols.

9.24

Colony PCR

Six colonies from the plate of electroporation no.1 and five from no.2 were chosen for colony PCR.

pPilin verification
Component	Volume/Details
2 × Taq Master Mix	25μL
ddH2O	20μL
GFR+GFF(5μM)	4μL
colony	1

The result is as follows. Two strains(designated as V.CR-cut and V.CR-mod respectively) were chosen to perform the following experiments.

Spread plate

ZIWEN XU

Overnight V.PC1 culture was spread out on twenty agar plates supplement with kanacymin, ampicillin, 0.5% glycerol and 2% arabinose. 300μL of culture was spread for each plate.The plates were cultured at 37℃ for 24 h before pilus harvest.

electrocompetent cell prepareation (protocol 2)

YUCHENG ZHANG

Electroporation competent ATCC14048 were prepared according to the protocols.

9.25

Electroporation

XIRAN HU

no.	Cell type	plasmid	Plasmid volume	voltage	time
1	ATCC14048	pGFPcut	5 μL	790 v	3 ms
2	ATCC14048	pGFPmod2	5 μL	790 v	3 ms

Green fluorescence measurement

XIRAN HU

Overnight culture of V.CR-cut and V.CR were used for green fluorescence measurement with microplate reader.The excitation light was 488 nm and emitted light was 535 nm. The result was as follows.

9.26

Pilus harvest

XIRAN HU

The cells were harvested from the plates with 1 mL of LB+v2 salts medium for each plate.The following procedures were the same as the previous harvest protocol.The BCA test result was as follows.The concentration of harvested pilin solution was 4.42 μg/μL.

Sample preparation for TEM

XIRAN HU

The pilin solution was seriously diluted. 5 μL of each sample was dropped on 200-mesh carbon-coated copper

grids and let air-dried for 2 h.Then the samples were stained by uranium acetate and dried at 4 ℃ overnight.

Colony PCR

XIRAN HU

Five colonies from the plate of electroporation no.1 and two from no.2 were chosen for colony PCR.

The result is as follows. Two strains(designated as V.cut and V.mod respectively) were chosen to perform the following experiments.

9.27

YICHEN LIU, YUCHENG ZHANG

Transmission electron microscopy

The result of TEM was as follows.There was too much impurity in our sample, so we could not acquire the structure of our pilus film.

9.28

Spread plate

ZIWEN XU

Overnight V.PC1 culture was spread out on twenty agar plates supplement with kanacymin, ampicillin, 0.5% glycerol and 2% arabinose. 300μL of culture was spread for each plate.The plates were cultured at 30℃ for 24 h before pilus harvest.

9.29

Spread plate

ZIWEN XU

Overnight culture of V.cut and V.mod was spread out on agar plates supplement with Ampicillin and cultured for 24 h before harvest to test the fluorscence intensity.

9.30

Pilus harvest and purification

XIRAN HU, ZIWEN XU

The cells were harvested from the plates with 1 mL of LB+v2 salts medium for each plate.The following procedures were the same as the previous harvest protocol(protocol 2). The harvested pilus solution was purified by Ni-NTA resin.

SDS-PAGE of pilin

ZIWEN XU

15 μL of washing 4 and elution 1 and elution 2 were loaded for SDS-PAGE. No obvious bands could be seen , maybe because the duratio of second ammonium sulfate precipiation was too short.

pdCas9-sg modification

YUCHENG ZHANG

Component	Volume/Concentration
2 × Phanta Flash Master Mix	25uL
ddH2O	20uL
repF+R(5 μM)	4uL
pdCas9-sg	1uL
annealing temperature	59℃
elongation time	40 sec

Gel extraction

Linearized pGFPmod1 was purified from PCR mix.

Gibson assembly

Component	Volume/Concentration
Linearized pdCas9-sg	2 μL(256ng)
2×CE mix	5uL
ddH2O	3uL

The recombinant products (desingated as pdCas9-sg2, pdCas9-sg3 and pdCas9-sg4) were transformed into chemical competent DH5α cells according to the protocol.

10.1

Colony PCR

XIRAN HU

Colonies from the plates of DH5α transfromed with modified pdCas9-ga were chosen for colony PCR. The result was as follows.One colony from each plate(designated as E.sg2, E.sg3 and E.sg4 respectively.) were chosen for the following experiments.

pPilin verification	Volume/Concentration
2 × Taq Master Mix	25uL
ddH2O	20uL
CRR+CRF(5uM)	4uL
colony	1

Spread plates

ZIWEN XU

Overnight V.PC1 culture was spread out on twenty agar plates supplement with kanacymin, ampicillin, 0.5% glycerol and 2% arabinose. 300uL of culture was spread for each plate.The plates were cultured at 30℃ for 48 h before pilus harvest.

10.3

Pilus harvest and purification

XIRAN HU

The cells were harvested from the plates with 1 mL of LB+v2 salts medium for each plate.The following procedures were the same as the previous harvest protocol(protocol 2). The harvested pilus solution was purified by Ni-NTA resin.

10.6

SDS-PAGE of pili

XIRAN HU

10 μL of all washing and elution solution were loaded for SDS-PAGE. The result was as follows.