iGEM 2023 — Team tongji-china

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![logo](https://static.igem.wiki/teams/4786/wiki/img/sidenav-logo.svg)

## Notebooks

[Week 1](#01)
[Week 2](#02)
[Week 3](#03)
[Week 4](#04)
[Week 5](#05)
[Week 6](#06)
[Week 7](#07)
[Week 8](#08)
[Week 9](#09)
[Week 10](#10)
[Week 11](#11)
[Week 12](#12)
[Back to top ⬆](#overview)

![overview_bg](https://static.igem.wiki/teams/4786/wiki/img/myoverview.png)

# Notebooks

![](https://static.igem.wiki/teams/4786/wiki/img/overview.png)

## Introduction

On this page, all experiments performed in our wet lab are recorded by date. Detailed experiment protocols are provided in the[experiment page](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D) and are therefore not described in this page

week-1 7.24~7.30

## 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:

![](https://static.igem.wiki/teams/4786/wiki/img/notebook/figure-7-28.png)

week-2 7.31~8.06

## 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.

![](https://static.igem.wiki/teams/4786/wiki/img/notebook/figure-8-6-1.jpg)

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

![](https://static.igem.wiki/teams/4786/wiki/img/notebook/figure-8-6-2.jpg)

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 |

week-3 8.07~8.13

## 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

![](https://static.igem.wiki/teams/4786/wiki/img/notebook/figure-8-7-1.png)![](https://static.igem.wiki/teams/4786/wiki/img/notebook/figure-8-7-2.png)

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 |

week-4 8.14~8.20

## 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.

![](https://static.igem.wiki/teams/4786/wiki/img/notebook/figure-8-18.png)

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.

![](https://static.igem.wiki/teams/4786/wiki/img/notebook/figure-8-20-1jpg.jpg)![](https://static.igem.wiki/teams/4786/wiki/img/notebook/figure-8-20-2.jpg)

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

week-5 8.21~8.27

## 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

![](https://static.igem.wiki/teams/4786/wiki/img/notebook/figure-8-25.jpg)

### 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.

![](https://static.igem.wiki/teams/4786/wiki/img/notebook/figure-8-26.jpg)

## 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 |

week-6 8.28~9.3

## 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:

![](https://static.igem.wiki/teams/4786/wiki/img/notebook/figure-8-28.jpg)

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.

![](https://static.igem.wiki/teams/4786/wiki/img/notebook/figure-8-28-2.jpg)

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.

week-7 9.4~9.10

## 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.

![](https://static.igem.wiki/teams/4786/wiki/img/notebook/figure-9-8.jpg)

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.

![](https://static.igem.wiki/teams/4786/wiki/img/notebook/figure-9-9.jpg)

## 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.

![](https://static.igem.wiki/teams/4786/wiki/img/notebook/fighre-9-10-1.jpg)![](https://static.igem.wiki/teams/4786/wiki/img/notebook/figure-9-10-2.jpg)

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℃ |

week-8 9.11~9.17

## 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.

![](https://static.igem.wiki/teams/4786/wiki/img/notebook/figure-9-11.png)

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.

![](https://static.igem.wiki/teams/4786/wiki/img/notebook/figure-9-12.jpg)

### 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.

![](https://static.igem.wiki/teams/4786/wiki/img/notebook/figure-9-13.png)

### 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.

![](https://static.igem.wiki/teams/4786/wiki/img/notebook/figure-9-15.jpg)

## 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.

![](https://static.igem.wiki/teams/4786/wiki/img/notebook/figure-9-16.png)

### 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.

![](https://static.igem.wiki/teams/4786/wiki/img/notebook/figure-9-17.png)

### 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℃ |

week-9 9.18~9.24

## 9.18

### WB of pilus

XIRAN HU

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

![](https://static.igem.wiki/teams/4786/wiki/img/notebook/figure-9-18-1.png)

### 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.

![](https://static.igem.wiki/teams/4786/wiki/img/notebook/figure-9-19.png)

### 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.

![](https://static.igem.wiki/teams/4786/wiki/img/notebook/figure-9-19-2.png)

### 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.

![](https://static.igem.wiki/teams/4786/wiki/img/notebook/figure-9-21.png)

### 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

![](https://static.igem.wiki/teams/4786/wiki/img/notebook/figure-9-22.png)

## 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.

week-10 9.25~10.1

## 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.

![](https://static.igem.wiki/teams/4786/wiki/img/notebook/figure-9-25.png)
![](https://static.igem.wiki/teams/4786/wiki/img/notebook/figure-9-25-2.png)

## 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.

![](https://static.igem.wiki/teams/4786/wiki/img/notebook/figure-9-26.png)

### 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.

![](https://static.igem.wiki/teams/4786/wiki/img/notebook/figure-9-27.jpg)

## 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.

![](https://static.igem.wiki/teams/4786/wiki/img/notebook/figure-9-30-1.jpg)

### 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 |

![](https://static.igem.wiki/teams/4786/wiki/img/notebook/figure-10-1-1.png)

![](https://static.igem.wiki/teams/4786/wiki/img/notebook/figure-10-1-2.png)

![](https://static.igem.wiki/teams/4786/wiki/img/notebook/figure-10-1-3.png)

### 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.

week-11 10.2~10.8

## 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.

![](https://static.igem.wiki/teams/4786/wiki/img/notebook/figure-10-6.jpg)

week-12 10.9~10.12

## 10.9

### Electroporation

XIRAN HU

| number | 1 | 2 | 3 |
| --- | --- | --- | --- |
|  |  |  |  |
| --- | --- | --- | --- |
| Cell type | ATCC14048 | ATCC14048 | ATCC14048 |
| plasmid | pdCas9-sg3 | pdCas9-sg4 | pdCas9-sg1 |
| Plasmid volume | 5 μL | 5 μL | 5 μL |
| voltage | 800 V | 800 V | 800 V |
| time | 4 ms | 4 ms | 4 ms |

### Chemical competent DH5α preparation

YUCHENG ZHANG

Chemical competent DH5α was prepared according to the protocols.

### Conductance measurement

Electric resistance of pili solution and supplemented with different antibodies were measured with a
multimeter.While maintaining a relatively constant filament concentration, we introduced equal concentrations
of different antibodies (with antibody solution volume much smaller than the filament solution volume) into
the solution.The result was as follows.

![](https://static.igem.wiki/teams/4786/wiki/img/notebook/figure-10-9.jpg)

## 10.10

### Electrocompetent cell prepareation (protocol 2)

YUCHENG ZHANG

Electroporation competent ATCC14048 were prepared according to the protocols.

### Electroporation

XIRAN HU

| number | 1 | 2 | 3 | 4 |
| --- | --- | --- | --- | --- |
|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Cell type | V.mod | V.mod | V.mod | V.mod |
| plasmid | pdCas9-sg1 | pdCas9-sg2 | pdCas9-sg3 | pdCas9-sg3 |
| Plasmid volume | 5 μL | 5 μL | 5 μL | 5 μL |
| voltage | 800 V | 800 V | 800 V | 800 V |
| time | 4 ms | 4 ms | 4 ms | 4 ms |

## 10.11

### Colony PCR

YUCHENG ZHANG

Sevral colonies from the plates electroporation were chosen for colony PCR. The result was as follows .One
colony from each plate(designated as VCR1-mod, VCR2-mod, VCR3-mod and VCR4-mod respectively.) were chosen for
the following experiments.The result was as follows.

| pPilin verification | 2 × Taq Master Mix | 25 μL |
| --- | --- | --- |
|  |  |  |
| --- | --- | --- |
| ddH2O | 20 μL |  |
| CRR+CRF(5 μM) | 4 μL |  |
| colony | 1 |  |

### Whole cell lysates WB for Pilin identification

XIRAN HU

Overnight culture of V.PC was inoculated to the following culture mediums, 20 mL for each condition:

| Culturing time\induction concentration | 0 | 20 mM |
| --- | --- | --- |
|  |  |  |
| --- | --- | --- |
| 6 h | 1 | 2 |

The induction of pilin expression was performed when the OD600 reached 0.5 by arabinose solution. The
cultures were maintained at 37 ℃

The primary antibody was diluted at 1:5000 and the second antibody was diluted at 1:5000.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the
engineering design cycle.{% endblock %}
{% block page\_content %}

Sorry, your browser does not support inline SVG.
![logo](https://static.igem.wiki/teams/4786/wiki/img/sidenav-logo.svg)

## Engineering success

[Overview](#overview)
[Wet lab part](#01)
[Modification of sfGFP reporter system](#02)
[Pili harvest](#03)
[Hardware Part](#04)
[Reference](#05)
[Back to top ⬆](#overview)

![overview_bg](https://static.igem.wiki/teams/4786/wiki/img/myoverview.png)

# Engineering success

## Wet Lab Part

## Modification of sfGFP reporter system

In order to find out the sgRNA that can repress PilA expression in Vibrio natriegens best, we designed
a reporter system based on sfGFP. A perfectly working CRISPRi system can elevate the ratio of our target Geobacter
metallireducens pilus in the final product and simplify the harvest step.

为了找到抑制PilA表达效果最好的sgRNA,我们设计了一个基于sfGFP的报告系统。一个完美工作的CRISPRi系统能够提高我们的目标——金属还原地杆菌菌毛在产物中的占比并且简化菌毛收获步骤。

### Cycle 1 The first version of report plasmid pGFP

#### design

In the first reporter plasmid pGFP, the expression of sfGFP is controlled by the promoter of
V.natriegens PilA and a small part of PilA is merged to the N terminus of sfGFP(fig.1). The target sequences of
the four candidate sgRNAs we initially chose reside in this region.

在第一个报告质粒pGFP中，sfGFP的表达由需钠弧菌PilA基因的启动子控制，并且sfGFP的末端融合了一小段PilA序列（图1）。我们最初选择的四个候选sgRNA的目标序列都位于这个区域之内。

![](https://static.igem.wiki/teams/4786/wiki/img/engineering/engn1.png)

Fig 1 the first version of report plasmid

#### Build

pGFP was ordered from GENWIZ. The structure of the plasmid is illustrated below(fig.2)

pGFP质粒是从GENWIZ订购的。该质粒的结构如下所示（图2）。

![](https://static.igem.wiki/teams/4786/wiki/img/engineering/engn2.png)

Fig 2 Basic structure of the first version of report plasmid.

#### test

We tried to measure fluorescence intensity using a microplate reader, but we failed to detect
difference between the transferomed and not transformed V.natriegens. Then we tried SDS-PAGE to check whether the
sfGFP was successfully experssed. There was a band between 35kD and 25kD(the molecular weight of recombined sfGFP
is about 30kD) in the lane of soluable proteins extracted from V.GF4(ATCC 14048 transfomred with pGFP) and not in
the lane of proteins from the wild type strain(fig.3). The SDS-PAGE was repeated three times to validate the
result.

我们尝试使用酶标仪来测量荧光强度，但在转化和未经转化的需钠弧菌中并没有检测到荧光强度的差异。之后我们尝试用SDS-PAGE来检查dfGFP是否成功表达。从V.GF4（转入sfGFP的ATCC
14048菌株）中提取的可溶性蛋白经电泳后在35kD和25kD之间有一个条带（重组sfGFP的分子量约为30kD），而野生型菌株对应的泳道中无相应条带。

![](https://static.igem.wiki/teams/4786/wiki/img/engineering/engn3.png)

Fig 3 SDS-PAGE result. The band of recombined sfGFP is marked with an
asterisk.(From left to right： V.GF4 total bacterial proteins, V.GF4 soluble proteins, wild-type sVibrio
natriegens total bacterial proteins, and wild-type Vibrio natriegens soluble proteins.)

#### learn

The result of SDS-PAGE showed that the recombinant sfGFP was expressed in V.GF4. But maybe due to the
PilA merged to the N terminus of sfGFP, it could not fold properly, so no fluorescence signal was detected.

SDS-PAGE的结果表明V.GF4中表达了重组sfGFP。但可能是由于sfGFP N末端融合的PilA片段，sfGFP无法正常折叠，因而无法检测到荧光信号。

After consulting professor Guo, we knew that the signal sequence in the merged part might interfere
with the folding of sfGFP because this sequence can interact with other proteins. Besides, sfGFP can fold properly
when the merged part is significantly larger or smaller than itself, or when there is a linker sequence between
the two parts. However, the lengths of the merged PilA and sfGFP were similar.

在请教郭教授后，我们了解到融合部分的信号序列会与其他蛋白质相互作用，因而可能会干扰sfGFP的折叠。此外，当添加的片段大小显著大于或小于sfGFP的大小时，或当被融合的两部分之间存在连接序列时，sfGFP也能够正常折叠。然而，我们所添加的PilA片段的长度与sfGFP近似。

### Cycle 2 The second version of reporter plasmid

#### design

In consideration of the possible problems we identified, we designed a second version of reporter
plasmid. We planned to delete the PilA part in the reporter plasmid so that there remained only two sgRNA target
sites in this version of plasmid(fig.4).

考虑到已被发现的一些可能的问题，我们设计了第二版报告质粒。我们计划删除报告质粒中的PilA片段，所以在这版质粒中知保留了两个sgRNA位点。

![](https://static.igem.wiki/teams/4786/wiki/img/engineering/engn4.png)

Fig 4 The second version of report plasmid,pGFPcut.

#### Build

We plan to modificate pGFP to get the new version of reporter plasmid, pGFPcut, by reverse PCR and
Gibson assembly(fig.5). Homologous arm was added to one of the primers for reverse PCR. The result plasmid pGFPcut
was first transformed into DH5α and verified by DNA sequencing.

我们计划通过反向PCR和Gibson
assembly来改进pGFP，得到新版的报告质粒pGFPcut。在其中一个引物上添加了同源臂以进行反向PCR。得到的pGFPcut质粒首先被转入DH5α中并通过DNA测序进行验证。

![](https://static.igem.wiki/teams/4786/wiki/img/engineering/engn5-1.png)
![](https://static.igem.wiki/teams/4786/wiki/img/engineering/engn5-2.png)
![](https://static.igem.wiki/teams/4786/wiki/img/engineering/engn5-3.png)

Fig 5 construction of pGFPcut.

#### learn

Although pGFPcut can express properly folded sfGFP as a reporter gene in V.natriegens and contains two
sgRNA target sites, we still wanted to test the other two sgRNAs.

尽管pGFPcut能够在需钠弧菌中表达正确折叠的sfGFP作为报告基因，并且包含了两个sgRNA位点，我们仍然想要进一步测试另外两个sgRNA。

### cycle 3 The third version of report plasmid

#### design

In order to test the other two sgRNAs, we decided to further improve the reporter plasmid. Based on
the first version, pGFP, we plan to alter the position of the initiation coden so that the PilA part could not be
translated(fig.6). In this new version, the three sgRNAs can be more precisely tested.

为了测试另外两个sgRNA，我们决定进一步改进报告质粒。基于第一版质粒pGFP，我们计划改变起始密码子的位置，使得PilA片段无法被翻译。在这一新版本中，候选的三种sgRNA可被更加精确地测试。

![](https://static.igem.wiki/teams/4786/wiki/img/engineering/engn6.png)

Fig 6 The third version of report plasmid,pGFPmod2.

#### Build

This time the modification was also based on mutagenesis PCR and Gibson assembly(fig.7). We used two
pairs of primers to change the two methionines to threonine. The resulting plasmid pGFPmod1 and pGFPmod2 were
verified by DNA sequencing.

这一次的改进也是基于定点突变PCR和Gibson assembly。我们使用两对引物分别将两个甲硫氨酸突变为苏氨酸。得到的质粒pGFPmod1和pGFPmod2通过DNA测序进行验证。

![](https://static.igem.wiki/teams/4786/wiki/img/engineering/engn-7.png)

Fig 7 Construction of pGFPmod2.

#### learn

pGFmod2 was successfully transfered into wild type Vibrio natriegens as verified by colony PCR.
V.natriegens transformed with pGFPmod2 and pGFPcut were named as V.mod and V.cut respectively. The fluorescence
intensity was measured using microplate reader and the result was illustrated below(fig.8). Three parallel tests
were conducted for the three strains and the average index of blank control(LB+v2 salts only) was deducted from
original data. The result showed that V.mod and V.cut had stronger fluorescence than the wild type strain.

pGFmod2被成功转入野生型需钠弧菌中并且经菌落PCR验证。将转入pGFPmod2和pGFPcut的需钠弧菌分别命名为V.mod和V.cut。用酶标仪测定荧光强度，结果如下（图8）。三个菌株各进行了三组平行实验。空白对照（LB+v2
salts）的平均值由原始数据推出。结果显示V.mod和V.cut的荧光强度强于野生型。

![](https://static.igem.wiki/teams/4786/wiki/img/engineering/gfp.png)

Fig 8 Fluorescence intensity measurement of two modified report plasmids.

## Pili harvest

### Cycle 1 shear and filter

#### design

In the early stage of our project design, we planned to use the method of shearing by blender and
filteration to harvest e-pili(fig.9). This method of pili harvest was originally used to harvest pili of Geobactor
sulfurreducens and two previous iGEM teams(GreatBay\_SZ 2020 and LINKS\_China 2020) used this method before.

在项目设计的早期，我们计划使用搅拌机搅碎并过滤的方式来收获e-pili（图9）。这种菌毛收获方法最初被用于收获硫地还原杆菌的菌毛。先前有两支iGEM队伍(GreatBay\_SZ 2020
and LINKS\_China 2020) 使用过该种方法。

![](https://static.igem.wiki/teams/4786/wiki/img/design/design13.png)

Fig 9 Initial workflow of pili harvest.

#### Test

We tried to harvest our e-pili according to this protocol see experiment. Generally, the cells were
scraped from plates and collected with LB+v2 salts medium and then pelleted by centrifugations and resuspended in
ethanolamine buffer. The mixture was blended to shear pili from cell surfaces and cell debris were removed from
the mixture. Finally, the solution was filtered with a 100 kDa-molecular-weight cutoff PVDF membrane. However,
when testing the resistance of protein membrane made of harvested sample solution, we found that the resistance
was too high(about 0.7 MΩ). So we tried transmission electron microscope to see microstructure of this solution
and the result was shown in fig.10. There were many thick fibers in the sample which were not e-pili.

我们尝试使用以下实验步骤来收获e-pili。简单来说，就是从平板上刮下细菌，收集在LB+v2
salt培养基中。之后离心，去上清，并用乙醇胺缓冲液重悬沉淀。得到的混合物加入搅拌机中搅拌，菌毛从细胞表面被剪切下来，细胞碎片被除去。最后，用PVDF-100kDa超滤膜过滤溶液。然而在测试由收获的样品溶液制成的蛋白质膜的电阻时，我们发现其电阻过高（约0.7
MΩ）。所以我们用透射电子显微镜对溶液的显微结构进行了观察，结果如图10所示。在样品中可见很多并非e-pili的粗纤维。

![](https://static.igem.wiki/teams/4786/wiki/img/engineering/gfp.png)

Fig 10 TEM result of pili harvested by the first method.

#### learn

We inferred that these fibers may be scrapped from PVDF membrane surface when collecting e-pili in the
final step of pili harvest. However, this protocol of pili harvest had been used by other investigators[1] and
iGEM teams. The problem may be caused by the difference in filter membrane brands and we might wash the membrane
too many times at the final step.

我们推测这些纤维可能是在收获菌毛的最后一步中从PVFD膜表面上被刮下的。然而，这一收获菌毛的实验步骤曾成功地被其他研究者和iGEM队伍使用过。问题可能出在超滤膜品牌的不同，也可能是由于我们在最后一步中将滤膜清洗了太多次。

### Cycle 2 shear and precipitate

#### design

After reading more relevant articles, we found another method to harvest pili. This method was similar
to the first one, but after shearing, cell debris was removed from the solution by centrifugation. Then, e-pili
was collected by ammonium sulfate precipitation(fig.11).

在阅读了更多相关文章后，我们找到了另一种收获菌毛的方法。这一方法与之前的类似，不同之处在于搅碎之后的细胞碎片通过离心从溶液中被移除。然后再通过硫酸铵沉淀法收集e-pili。

![](https://static.igem.wiki/teams/4786/wiki/img/engineering/engn-11.png)

Fig 11 Modified workflow of pili harvest.

#### test

TEM and resistance measurement were also performed using purified pili solution harvested using this
method. This time the resistance of the protein membrane was much lower, which was about 17 kΩ. The result of TEM
showed that there were several nanowires in the solution, which we inferred to be the e-pili we wanted.

对用这种方法收获的菌毛溶液也进行了电镜观察和电阻测量。这一次蛋白质膜的电阻与之前相比显著降低，约为17
kΩ。电镜观察结果显示，溶液中存在着一些纳米线，我们推测这些纳米线就是我们想要得到的e-pili 。

![](https://static.igem.wiki/teams/4786/wiki/img/engineering/engn-12.jpg)

Fig 12 TEM result of pili harvested by the second method.

#### learn

Using the second method, we successfully acquired pili slution that had much lower resistance than
that of 5% NaCl solution(see [result](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) ). However, the
resistance of this pili was still higher than formerly
reported[2]. The reason for this result might be that there were still many impurities in our sample due to
insufficient speed provided by our centrifuge when removing debris from the pili-solution. Also, we are not
skilled in purification with Ni-NTA resin and sample preparation for TEM.

使用第二种收获方法，我们成功得到了电阻率显著低于5%
氯化钠溶液的菌毛溶液。然而，该菌毛溶液的电阻仍高于先前的相关报告。这一结果的原因可能是：将细胞碎片菌毛溶液中去除时，由于我们的离心机转速不够，导致我们的最终样品中仍然存在很多杂质。同时我们对于使用Ni-NTA树脂纯化蛋白和准备电镜观察样品也不够精通。

## Hardware Part

### Bio-degradable Electrode for E-Pili Cartridge

As E-Pili Cartridge is produced, used and discarded by the millions, and its use is no longer limited
to healthcare facilities, we believe it is important to minimize the use or percentage of unsustainable components
in this consumable. Considering the SDGs of poverty eradication and responsible consumption and production: we
believe that the use of unsustainable components or the percentage of such components in this consumable should be
minimized in order to reduce the damage to the environment throughout the entire life cycle of the consumable. And
to provide the lowest possible cost options for E-Pili Cartridge to make them affordable to a wider range of
potential users (outside of urban community health systems).

由于E-Pili
Cartridge会数以千万记地被生产、使用和丢弃以及使用场景不再局限在医疗机构内，考虑到可持续发展目标中消除贫困和负责任地消费和生产：我们认为应当尽可能减少这种耗材中不可持续的成分使用或该成分的占比，以减少耗材的全生命周期对环境的损害；并提供尽可能低成本的耗材方案，以让更多的潜在用户（城市社区卫生体系以外）得以负担得起。

#### design

Structures in the conventional lateral flow immunochromatographic assay include: NC membrane,
absorbent pad, sample pad, PVC base plate and casing, to which our design adds E-Pili and electrodes. The parts of
the Cartridge that are difficult to degrade are the electrodes, the PVC base plate and the casing. Since the rapid
test consumables with electrodes are the more innovative part of our design, we wanted to focus on responsible
production by replacing the more commonly used copper electrodes with biodegradable electrode materials; there are
already examples of pulp-based materials used in the base plate and casing, so we are not exploring this part of
the design for the time being.

常规的侧流免疫层析测定方式中的结构包括：NC膜、吸水垫、样品垫、PVC底板和外壳，我们的设计在其中添加了菌毛和电极。耗材中难以降解的部分有电极、PVC底板和外壳。由于带有电极的快速检测耗材是我们设计中较为创新的部分，我们希望将负责任的生产集中落实于此，用可降解的电极材料替换更常用的铜电极；底板与外壳已有纸浆基材料制造的案例可参考，故我们暂不在这个部分做探索。

During our research we found a HCI paper that describes sustainable electronic prototyping with
bio-based and biodegradable materials, providing formulations for conductive bioplastics in the form of sheets,
pastes and foams. We attempted to explore molding methods using the sheet and paste forms of this conductive
bioplastic as materials to be used as electrodes for the E-Pili Cartridge.

在调研中我们发现了一篇人机交互论文，论文介绍了用以生物基且可生物降解的材料进行可持续电子原型制作方式，提供了片材、糊剂和泡沫三种形式的导电生物塑料的配方。我们试图用这种导电生物塑料的片材和糊剂形式为材料，探索成型方式，将其作为E-Pili
Cartridge的电极。

#### Build

![](https://static.igem.wiki/teams/4786/wiki/img/engineering/fig1.jpeg)

Fig.13 Raw Materials and Tools for Material Testing

We experimented with electrode materials at Fablab Shanghai and tried both sheet and paste forms of
them as fabricated in the paper. The Carboxymethyl Cellulose (CMC) based paste was very prone to drying and
cracking and could not be molded, so no further attempts were made.

我们在Fablab Shanghai进行电极材料的实验，并按照论文中的制作方式尝试了其中的片材和糊剂两种形式，其中羧甲基纤维素（CMC）为基材的糊剂十分容易干裂，无法成型，故未做进一步尝试。

Sheets were made with 3g of sodium alginate as a base, mixed with 75g of distilled water and 3ml
(3.8g) of glycerol was added. After standing for two hours, 1.95g of carbon black powder was added and stirred.
The mixture was applied to an acrylic plate (the plate brush was set up on a 2mm thick strip of cork to control
the thickness), to which a 10% calcium acetate solution was sprayed. It was then dried intermittently in an oven
at 50°C for two days (with the oven door slightly open to prevent overheating and warping).

片材以3g海藻酸钠为基底，与75g蒸馏水混合，加入3ml（3.8g）甘油。静置两小时后，加入1.95g炭黑粉末搅匀。将混合物涂布在亚克力板上（将板刷架在2mm厚的软木条上以控制厚度），向其上喷洒10%醋酸钙溶液。随后放入烤箱50℃间断地烘干两天（烤箱门稍开，防止过热翘边）。

![](https://static.igem.wiki/teams/4786/wiki/img/engineering/fig2.jpeg)

Fig.14 Oven Drying

![](https://static.igem.wiki/teams/4786/wiki/img/engineering/fig3.png)

Fig.15 Biodegradable Conductive Plastic Sheet

#### test

A variety of ratios and drying methods were tried and documented. The resistance of the sheet with
sodium alginate as the base material and carbon black as the conductor was large (on the order of 10 kΩ in the
initial experiment), but after raising the proportion of carbon black (3.5 g) and reducing the proportion of
glycerol (3.5 g), the smallest portion of the sheet resistance (with the meter pens spaced at 1 cm apart) could be
controlled to be in the range of 20-60Ω.

我们进行了多种配比和干燥方式的尝试，并将其记录下来。海藻酸钠为基材、炭黑为导电成分的片材电阻较大（初次实验为10kΩ数量级），但在提升炭黑比例（3.5g）并减少甘油比例（3.5g）后，片材电阻最小的部分（表笔间距1cm)可以控制在20-60Ω。

![](https://static.igem.wiki/teams/4786/wiki/img/engineering/fig4.png)

Fig.16 Experimental Records of Different Batches

#### learn

We obtained materials with more ideal resistance values by changing the material ratio, but the way of
producing such bio-based electrodes in Fablab is rather rough. Under the current experimental conditions,
different batches of materials are affected by humidity, area, substrate/mold, etc., and the resistance and
flatness of the output materials are not guaranteed to be stable. We tried to compensate for the effect of
electrode instability by first measuring the electrode resistance, then the resistance of electrode + E-Pili, and
finally calculating the difference to get the resistance of E-Pili. The distribution of resistance in a piece of
material was also not uniform. It is noteworthy that we found that the resistance of the part of the material that
was tightly attached to the acrylic plate after drying and smooth (visibly reflective) when removed was very small
(around 20 Ω), whereas the resistance of the large, rough, and dry part was still very large (on the order of
100-1000 Ω).

我们通过改变材料比例获得了电阻值较为理想的材料，但在Fablab中生产这种生物基电极的方式比较粗糙，目前实验条件下，不同批次材料受湿度、面积、基底/模具等因素的影响较大，产出材料的电阻、平整程度都无法保证稳定。我们试图用先测定电极电阻，再测电极+菌毛的电阻，最后计算差值得到菌毛电阻的方式来补偿电极不稳定的影响。一块材料中的电阻分布也并不均匀。值得注意的是我们发现烘干后紧贴亚克力板，摘下后光滑（明显反光）的部分电阻很小（20Ω左右），而大面积的粗糙且干燥的部分电阻仍很大（100-1000Ω数量级）。

Another issue of great impact is that this sheet material cannot be manufactured as finely as metal
for the time being in the form of interdigital electrodes, nor can it be molded into cylinders to serve as
contacts between the Cartridge and Plinker.

另一个影响很大的问题是这种片状的材料暂时无法像金属一样很精细地加工为叉指电极的形式，也很难成型为圆柱体作为耗材和设备之间的触点。

The question of how to process this material so that it can be used as our electrodes is an open one,
but we have had to put the exploration of sustainable electrodes on hold due to the project timeline. However, we
will be actively exploring ways in which it can be stabilized and manufactured in a way that makes our Cartridge
environmentally friendly.

对于这种材料如何加工以使其可以用作我们的电极是一个有待解决的问题，但由于项目周期问题我们不得不搁置对于可持续电极的探索。但我们会积极探索使其可以被稳定制造的方式，让我们的耗材对环境友好。

### Engineering cycle

### cycle1

#### Design

During the initial debugging phase, we conducted a study on the expansion of WiFi functionality for the STM32. The basic process for testing the WiFi functionality of the STM32 chip includes importing relevant serial port libraries, using the WiFi module for communication tests to achieve data reception and transmission.

#### Build

We performed WiFi debugging using the STM32 chip. Firstly, we imported the serial port library "serial" in Python to read the serial port data. Upon successful reading, since the transmitted signal is in the form of a floating-point number with 8 significant digits, we needed to convert the string data into an integer and set the decimal places accordingly.

![](https://static.igem.wiki/teams/4786/wiki/img/engineering/e1.png)

#### Test

We conducted communication testing using the WiFi module of the STM32 chip. First, we ensured the WiFi module was properly connected to the WiFi network we set up and connected it based on the account and password we set. Then, we sent WiFi connection instructions to the WiFi module via serial communication to ensure connection with the WiFi network. If the connection was successful, the WiFi module would return connection status information and we could use it for data transmission.

![](https://static.igem.wiki/teams/4786/wiki/img/engineering/e2.png)

#### Learn

Through this study, we learned the basic process and debugging methods of using the WiFi function of the STM32 chip. We mastered skills such as serial data reading and sending connection instructions to the WiFi module. At the same time, we also deepened our understanding of the WiFi function in chip design, which is very helpful for subsequent device design and development.

### cycle2

#### Design

Through this study, we learned the basic process and debugging methods of using the WiFi function of the STM32 chip. We mastered skills such as serial data reading and sending connection instructions to the WiFi module. At the same time, we also deepened our understanding of the WiFi function in chip design, which is very helpful for subsequent device design and development.

#### Build

We establish a real-time curve plotting model in the form of dynamically displaying a conductivity curve chart. In Python, we use library functions for graphical plotting. Firstly, we use the functions under the library to set the initial set and use pyqtgraph to draw the initial curve. Once started, the serial port begins receiving data. Each time new data is received, it is added directly to the set, and the entire curve is updated simultaneously, achieving real-time curve plotting. In order to achieve the effect of real-time display, after each new data is added to the end of the set, the overall image needs to be shifted left by one data, that is, the beginning data in the set is deleted, and the entire curve is redrawn.

#### Test

![](https://static.igem.wiki/teams/4786/wiki/img/engineering/e3.png)

#### Learn

We learned how to use Python to realize real-time curve plotting of data, as well as the skill of connecting with STM32 and reading data. We mastered the basic process of using the matplotlib library and pyqtgraph library to plot curves, and understood the importance of filtering and data processing. In addition, we also learned how to set appropriate parameters and achieve real-time plotting to ensure the accuracy and readability of curve plotting.

### cycle3

#### Design

Finally, we debugged the STC15 chip and ESP8266 WiFi module, which are closer to the functionality of conductivity testing, for hardware connection. We used serial communication functions to communicate and configure the ESP8266 module, and ultimately ensured that the ESP8266 module can communicate normally between the client and server side.

#### Build

First, connect the hardware equipment, including the ESP8266 WiFi module, STC15 chip development board, and module for testing data. Connect the TXD pin of the ESP8266 module to the RXD pin of the STC15 development board, and connect the RXD pin of the module to the TXD pin of the development board. Also, connect the VCC pin to the 5V power pin on the development board and the GND pin to the GND pin on the board. Next, use a USB to TTL serial module to connect the STC15 development board to the computer.

During serial communication, use the sending and receiving commands in the STC15 development environment to communicate with the ESP8266 module. After setting the AT command, send the AT command through UART to set up the ESP8266 module, including checking its normal operation and setting up WiFi connection parameters. To ensure that the ESP8266 module works properly, write code using serial communication functions.

![](https://static.igem.wiki/teams/4786/wiki/img/engineering/e4.png)

During serial communication, use the sending and receiving commands in the STC15 development environment to communicate with the ESP8266 module. After setting the AT command, send the AT command through UART to set up the ESP8266 module, including checking its normal operation and setting up WiFi connection parameters. To ensure that the ESP8266 module works properly, write code using serial communication functions.

#### Test

When conducting client testing, the first step is to download a TCP client software and enter the IP address and port number of the ESP8266 module in the software for connection testing and message sending and receiving testing. Throughout the testing process, it is important to ensure that the network environment is stable and that the WiFi name and password set in the software are correctly connected. Before starting the test, check the connection status of the ESP8266 module, such as the flashing of the indicator lights, to ensure that a proper connection can be established. During testing, it is necessary to pay attention to the stability of the communication and to conduct a long-term testing to ensure stable data transmission. By conducting client testing, it further verifies that the STC15 chip and ESP8266 module can work properly and ensure smooth communication under all circumstances.

![](https://static.igem.wiki/teams/4786/wiki/img/engineering/e5.png)

#### Learn

We have learned how to debug hardware devices, communicate using serial communication functions, and perform client testing. We also learned about the connection methods between the STC15 chip and the ESP8266 WiFi module, which can improve performance in applications like conductivity testing. In addition, we learned how to ensure network stability and reliable data transmission for more reliable communication in practical applications.

References

[1]Ueki T, Walker DJF, Woodard TL, Nevin KP, Nonnenmann SS, Lovley DR. An Escherichia coli Chassis for
Production of Electrically Conductive Protein Nanowires. ACS Synth Biol. 2020 Mar 20;9(3):647-654.

[2]Tan Y, Adhikari RY, Malvankar NS, Ward JE, Woodard TL, Nevin KP, Lovley DR. Expressing the Geobacter
metallireducens PilA in Geobacter sulfurreducens Yields Pili with Exceptional Conductivity. mBio. 2017 Jan
17;8(1):e02203-16.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

Sorry, your browser does not support inline SVG.
![logo](https://static.igem.wiki/teams/4786/wiki/img/sidenav-logo.svg)

## Experiments

[Chemical competent Vibrio natriegens preparation](#01)
[Chemical competent Vibrio natriegens tansformation](#02)
[Chemical conpetent DH5α preparation](#03)
[Chemical competent DH5α transformation](#04)
[Electrocompetent Vibrio natriegens preparation](#05)
[Electroporation](#06)
[Colony PCR](#07)
[Pilin expression](#08)
[SpdCas9 expression](#09)
[Pilus harvest](#10)
[SDS-PAGE analysis](#11)
[Western blot analysis of pilin](#12)
[Gibson assembly](#13)
[Gel DNA extraction](#14)
[Supplement note](#15)
[Back to top ⬆](#overview)

![overview_bg](https://static.igem.wiki/teams/4786/wiki/img/myoverview.png)

# Experiments

![](https://static.igem.wiki/teams/4786/wiki/img/overview.png)

## Introduction

On this page ,we list detailed descriptions about all experiments we carried out during our
project and some supplement information.We hope that these protocols and information could be of
some help to other teams.

Chemical competent *Vibrio natriegens* preparation

1. 150 mL of BHI + v2 salts(supplement 1) is inoculated directly from
a glycerol stock of *V. natriegens* and incubated at 37 °C with
agitation at 200 r.p.m. to an OD600 of 0.4 (~2 h).

(All subsequent steps are performed quickly at room temperature. )

2. The culture is split into three 50-mL conical tubes, and
centrifugated at 3,000 × g for 5 min.

3. Remove the supernatant carefully and gently suspend the
precipitation with 5 mL 100 mM MgCl2.

4. Consolidate the cells into two 50-mL conical tubes, and the volume
in each tube is brought up to 30 mL with 100 mM MgCl2 and mixed by
gentle inversion.

5. Centrifugation at 3,000 × g for 4 min.

6. Suspend the precipitation in 5 mL 100 mM CaCl2, consolidate them
into one tube, and then brought the volume up to 30 ml with additional
100 mM CaCl2.

7. Mix the tube gently by inversion and then incubate it at room
temperature for 20 min.

8. Centrifugation at 3,000 × g for 4 min.

9. Remove the supernatant, and the cells are resuspended in ~1.5 mL .
The cells are then aliquoted into chilled tubes, frozen in a dry ice
bath, and stored at −80 °C until use.

Chemical competent *Vibrio natriegens* tansformation

1. Take a vial of competent cells and thaw it on ice.

2. Add the plasmid into 50 μL of competent cells and gently mix them, then incubate on ice for
20–30 min.

(During incubation, 1 mL BHI + v2 salts without antibiotics is warmed to 37 °C in a water bath.)

3. The cells and DNA are incubated at 42 °C for 45 s in a water bath and returned to ice for 2
min.

4. 37 °C-warmed medium is used to transfer the cells out of the microcentrifuge tube and into the
culture tube.

5. Once transferred, the culture tube is maintained in a 37 °C water bath for 1 min and then
allowed to recover by being incubated at 37 °C for 2 h with an agitation of 200 rpm. Cell
dilutions are plated on warmed selective plates and incubated overnight at 37 °C.

Chemical conpetent DH5α preparation

1. Inoculate DH5α strain in LB liquid medium overnight;

2. Expanded inoculation in 3mL LB liquid medium at a ratio of 1:100 until OD600 ≈ 0.5 (about 2
hours);

3. Ice bath for 20 min;

4. Take 1mL of bacterial solution, centrifuge at 5000 rpm, 4℃ for 5min, repeat 3 times;

5. Discard the supernatant, carefully resuspend the bacterial precipitate with 1mL of 1M CaCl2
and incubate on ice for 30 min;

6. Centrifuge at 5000 rpm, centrifuge at 4℃ for 5min;

7. Discard the supernatant, carefully resuspend the bacterial precipitate with 200 μL 1M CaCl2,
equally divided into 2 centrifuge tubes, which can be stored at -80℃ by adding 50% glycerol at
1:1.

Chemical competent DH5α transformation

1. Take 100μl of the competent cell from -80℃ then thaw it on ice.

2. Add plasmid DNA solution (5μL about 600ng), mix gently and leave on
ice for 30 minutes.

3. Heat shock at 42℃ water bath for 90 seconds. After heat shock,
quickly put on ice to cool for 2 minutes.

4. Add 1ml of LB liquid medium (without antibiotics) into the tube,
mix and incubate at 37℃ for 1 hour to recover.

5. Cell dilutions are plated on warmed selective plates and incubated
overnight at 37 °C for the colonies to appear.

Electrocompetent *Vibrio natriegens* preparation

1. 100μL of seed solution was inoculated in 50mL LB+v2(supplement 1)
liquid medium and incubated at 37℃, 250RPM until OD600=0.4-0.6.

2. The bacterial solution was transferred to a pre-cooled 50mL
centrifuge tube and ice-bathed for 5 mins.

3. Centrifuge at 4℃, 2000RCF for 5 mins, and discard the supernatant.

4. The bacterial precipitate was gently resuspended in 1mL pre-cooled
1M sorbitol buffer.

5. Centrifuge at 5000 RCF for 5 mins at 4℃ and discard the
supernatant.

6. The bacterial precipitate was gently resuspended in 1mL pre-cooled
1M sorbitol buffer.

7. Centrifuge at 5000 RCF for 5 mins at 4℃, and discard the
supernatant.

8. The bacterial precipitate was gently resuspended in 250 μL
pre-cooled 1 M sorbitol buffer.

9. Dispense 50μL into each tube and store the tubes at -80℃ until use.

Electroporation

1. A vial of electrocompetent cell (50 μL) is retrieved from storage at
-80 °C and thawed on ice.

2. About 100ng plasmid is added into the cell and the 1.5 mL centrifuge
tube is incubated on ice for 3 mins.

3. The mix is gently transferred into chilled electroporation cuvette
with a 0.1-cm gap size and allowed to stand for three minutes.

4. Cells are electroporated at a voltage of 790v and a constant time of
4ms (Bio-Rad,MicroPulserTM).

5. The cells are immediately transferred into 1 mL chilled LB+v2 salts
medium and cultured at 37℃ with agitation at 250 r.p.m for 1 h.

6. 50 μL of the recovery culture is plated out on a warm agar plate with
appropriate antibiotic.

7. The plate is incubated at 37℃ overnight for the colonies to grow.

Colony PCR

1. Preparation of culture medium：

Add 2 mL LB+v2 salts medium with appropriate antibiotic (supplemet
note 2) into several 15 mL centrifuge tubes (according to the number
of colonies) and prewarm the medium at room temperature.

2. Preparation of PCR mix: for 50 μL PCR

| Component | Volume |
| --- | --- |
| 2×Taq Master Mix（dye plus） | 25 μL |
| ddH2O | 20 μL |
| Forward primer(10 μM) | 2 μL |
| Reversed primer(10 μM) | 2 μL |

3. A small colony is suspended in the PCR mix, and the pipette tip is
tipped into the medium with appropriate antibiotic.

4. Culture medium with the pipette tip is cultured at 37 ℃ with an
agitation of 210 r.p.m.

5. Set up the PCR program:

| Step | Temp(℃) | Time | Cycles |
| --- | --- | --- | --- |
| Initial denaturation | 95 | 3 min | 1 |
| denaturation | 95 | 15 sec | 20-35 |
| annealing | Tm-2 ℃ | 15 sec | 20-35 |
| extension | 72 | 60 sec/kb a | 20-35 |
| Final extension | 72 | 5 min | 1 |
| cooling | 4 | 20 min | 1 |

a: For 2× Phanta Flash Master Mix(Dye Plus), the extending speed is 5
sec/kb.

6. Choose appropriate culture according to the PCR result to conduct
the following experiments.

Pilin expression

1. 2 mL LB+v2 salts with an Ampicilin concentration of 100 μg/mL is
inoculated with V.natriegens containing appropriate plasmid and
cultured at 37℃ with an agitation of 210 r.p.m overnight.

2. 60 mL of LB+v2 salts with 100 μg/mL of Ampicilin is inoculated with
the overnight culture at a dilution of 1:100, and cultured at 37℃ with
an agitation of 210 r.p.m for about 1.5 h till its OD600 reached 0.5.

3. Arabinose storage solution (1 M) is added to the culture at a
dilution of 1:50 to induce the expression of pilin.

4. The culture is continuously incubated at 37 ℃ with an agitation of
210 r.p.m overnight to prepare for the harvest.

SpdCas9 expression

1. 2mL LB+v2 salts with 100μg/mL kanamycin is inoculated with V.natriegens containing appropriate
plasmids and cultured at 37℃ with an agitation of 210 r.p.m overnight.

2. 60 mL LB+v2 salts with 100μg/mL of kanamycin is inoculated with the overnight culture at a
dilution of 1:100, and cultured at 37℃ with an agitation of 210 r.p.m for about 1.5 h till its
OD600 reached 0.5.

3. IPTG storage solution (50 mg/ml) is added to the culture at a dilution of 1:100 to induce the
expression of SpdCas9.

4. The culture is continuously incubated at 37℃ with an agitation of 210 r.p.m for 5h to prepare
for the harvest.

Pilus harvest

1. 60 mL of overnight culture is centrifuged at 4000×g for 15 min at 4
°C.

2. The cells are resuspended in 30 mL 150 mM ethanolamine buffer (pH
10.5).

3. The suspension is transferred into a warring blender.

4. Wash the centrifuge tube with 20 mL 50 mM ethanolamine buffer(
which is also added to the blender) three times.

5. The 90 mL suspension was blended for 2 min at a low speed to shear
pilus from the cells.

6. The contents of the blender are transferred to a centrifuge bottle
along with a wash of the blender with 10mL of ethanolamine buffer.

7. The blended material is centrifuged at 5000×g for 30 min at 4 °C to
remove the cells.

8. The supernatant is collected and Triton X-100 detergent is added to
a final concentration of 6 mM to solubilize any remaining cellular
debris.

9. The tube is shaken at 100 r.p.m at room temperature for 45 min.

10. Contents in the tube are added to a stirring filtration unit that
has a membrane filter with a 100 kDa- molecular-weight cutoff .

11. Additional ethanolamine buffer is added to the mixture to yield a
final 2 mM concentration of Triton X-100.

12. The mixture is filtered in nitrogen gas (1.5MPa)

13. The pilins are collected from the filter membrane by scraping the
surface into 1mL water.

SDS-PAGE analysis

1. Sample preparation

a. Soluable bacterial native proteins:

extracted by Bacterial Active Protein Extraction Reagent.

b. Cell bodies:

1 mL of bacteria culture is transferred into a 1.5mL centrifuge tube
and cells are pelleted at 10000 r.p.m for 1 mins.The cell pellets then
are collected and resuspended with 80 μL ddH2O.

c. Purified pilus solution:

Pilus solution is prepared as described above.

2. protein loading buffer is added to the sample according to the
volume.a

3. Mix the contents by vortexing and contrifuge the tube to get the
contents at the bottom.

4. Heat the samples at 100 ℃ for 5 mins in metal bath to denature the
proteins.

5. Gel preparation:

Tris-Gly SDS-PAGE gels are prepared for SpdCas9 analysis. Tris-tricine
SDS-PAGE gels are prepared for pilin monomer analysis.

6. Load 10 µL of the prepared loading samples to the gel, and load 5 μL
of prestained protein marker to the gel.

7. Run the gel at 80 v for about 30 mins until the samples reach the
boundary between stacking gel and separating gel.

8. Run the gel at 150 v for about 60 mins until the Bromophenol blue
reaches the bottom of the gel.

9. Take the gel out of the casting frame and put it in a tray.

10. Add about 30 mL of ddH2O (accoring to the volume of the
tray. Make sure that the gel is completely submerged in ddH2O) and
heat in microwave till the water boils.

11. Put the tray on a shaker to incubate the gel for about 5 min.

12. Discard the water and add about 20mL of Coomassie brilliant blue R-250
solution into the tray, then put the tray on a shaker to incubate the
gel for about 10 min.

13. Discard the Coomassie brilliant blue R-250 solution and add 20 mL of
ddH2O. Put the tray on a shaker to incubate for about 10 min.

14. Discard ddH2O and add 20 mL of destaining
solution(supplement note 2) to wash the gel on a shaker 3 times(10
mins each time).

Western blot analysis of pilin

### SDS-PAGE

1. SDS-PAGE protocols are the same as previously described in steps 1-9
of SDS-PAGE analysis.

### wet transfer

1. Cut the upper right corner of the gel and PVDF membrane (0.22 μm) to
mark their direction.

2. Activate the PVDF membrane in methyl alcohol for 20 sec.

3. Make the “sandwich” as follows: cathode-sponge-filter paper-gel-filter
paper-sponge-canode. Keep the “sandwich” hydrated while making it to
avoid the formation of bubbles between the layers.

4. Assemble the device and transfer the proteins at 200mA for 50 mins

5. Carefully put the membrane in a clean tray (make sure that the side
containing proteins is upward) and stain the PVDF membrane in ponceau
S staining solution for 10 mins, then wash the membrane with TBST
several times until the protein bands appears.

### Blocking and antibody incubation

1. Add 30 mL 5% skim milk solution to the tray and incubate the membrane
on a shaker at room temperature for 1 h.

2. Discard the skim milk solution and wash the membrane with TBST three
times, for 5 mins each time.

3. Discard TBST and add 10 mL primary antibody solution(diluted with TBST
at 1:5000) to the tray, then incubate the membrane at 4 ℃ overnight.

4. Recycle the primary antibody solution and store it at 4 ℃.

5. Wash the membrane with TBST three times, for 5 mins each time.

6.
Discard TBST and add 10 mL secondary antibody solution(diluted with
TBST at 1:10000) to the tray, then incubate the membrane at room
temperature for 1.5 h.

7. Wash the membrane with TBST three times, for 5 mins each time.

### Image development (Chemiluminescence)

1. Mix equal volume of Luminol solution and eroxide solution to a total
volume of 1 mL.

2. Discard TBST in the tray and add the mixture to the surface of the
membrane.

3. Put the membrane into ChemiDoc MP system（Bio-rad）and develop the
image according to instructions from the manufacturer.

Gibson assembly

### Linearized plasmid self-association assembly

1. DNA fragment of the linearized plasmids is obtained by high-fidelity
PCR reaction (reverse PCR) with the addition of homology arms.

2. Prepare the following reaction system on ice (Using ClonExpress Ultra
One Step Cloning Kit V2):

| Component | Volume |
| --- | --- |
| Linearized vector fragment | 2x 1 μL (~2x100 ng) |
| 2×CE Mix | 5 μL |
| dd H2O | 3 μL |

3. Mix by gentle suction and beating with a pipette (do not shake to mix)
and briefly centrifuge to collect the reaction solution at the bottom
of the tube.

4. Incubate at 50°C for 5 mins; cool to 4°C or immediately put the tube
on ice.

5. Thaw the cloned chemoreceptor cells on ice.

6. Add 5 -10 μL recombinant product to 100 μL competent cells, flick the
wall of the tube to mix (do not oscillate to mix), and leave it on ice
for 30 mins.

7. After heat shock in 42℃ water bath for 30 sec, immediately put the
tube on ice to cool for 2 - 3 min.

8. Add 900 μl of LB liquid medium (without antibiotics) and shake the
bacteria at 37°C for 1 h (200 - 250 rpm).

9. Ampicillin-resistant LB solid medium plates were pre-warmed in a 37°C
incubator.

10. The plates were centrifuged at 5,000 rpm (2,500 × g) for 5 min and 900
μl of the supernatant was discarded. Bacteria were resuspended with
the remaining medium and gently spread with a sterile applicator stick
on plates containing ampicillin resistance.

11. The plates were inverted and incubated at 37°C for 12 - 16 h in an
incubator.

### BCA test

Concentration of pilA protein monomer was measured by 96-well ELISA
plate method using BCA Protein Quantification Kit.

1. BCA working solution preparation: according to the number of samples,
add 50 volumes of BCA Reagent A plus 1 volume of BCA Reagent B (50:1)
to prepare the appropriate amount of BCA working solution. Mix them
thoroughly.

2. Standard curve: Take a piece of ELISA plate and add reagents according
to the data in the table below:

| No. | 0 | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Protein standard solution (μL) | 0 | 1 | 2 | 4 | 8 | 12 | 16 | 20 |
| ddH2O (μL) | 20 | 19 | 18 | 16 | 12 | 8 | 4 | 0 |
| BCA working solution(μL) | 200 | 200 | 200 | 200 | 200 | 200 | 200 | 200 |
| Corresponding protein content (μg) | 0 | 1 | 2 | 4 | 8 | 12 | 16 | 20 |

3. Sample preparation: Dilute the protein sample to be measured with
ddH2O to the appropriate concentration, take 20 μl of the sample and
add 200 μl of BCA working solution.

4. After shaking and mixing, place the sample at 37℃ for 20 - 30 min.

5. Determine the absorbance value at A562 nm with an ELISA plate, and use
the absorbance value without BSA as a blank control.

6. Plot the standard curve with protein content(μg) as the horizontal
coordinate and absorbance value as the vertical coordinate.

7. According to the measured absorbance value, the protein content of the
sample can be calculated based on the standard curve.

8. Calculate the protein concentration: Divide the protein content by the
sample volume of 20 μl and multiply by the corresponding dilution
factor to obtain the actual concentration of the sample to be tested.

### Glycerol stock

1. Prepare an overnight culture of the strain to be stored with
appropriate liquid medium(OD600=1~1.5).

2. Add 500 μL of prepared culture to 500 μL of 50% glycerol.

3. Mix gently and store the tube at -80℃ until use.

Gel DNA extraction

### Gel Recovery Program

1. Cut off the gel containing the target DNA fragments. 100 mg of gel
equals 100 μL of volume as a gel volume.

2. Add an equal volume of Buffer GDP. 55°C water bath for 10 min. Invert
and mix during the water bath.

3. Centrifuge quickly and place the FastPure DNA Mini Columns-G in a
Collection Tubes 2 mL collection tube, transfer ≤700 μL of lysate to
the column, centrifuge at 12,000 rpm (13,800 × g) for 60 sec.

4. （If the volume of lysate is >700 μL, return the column to the
Collection Tubes, and repeat step 3）

5. Discard the filtrate. Add 300 μL Buffer GDP to the column. Centrifuge
at 12,000 rpm (13,800 × g) for 60 sec.

6. Discard the filtrate. Add 700 μL of Buffer GW (with anhydrous ethanol
added) to the column. Centrifuge at 12,000 rpm (13,800 × g) for 60
sec.

Repeat step 5.

7. Discard the filtrate. Centrifuge at 12,000 rpm (13,800 × g) for 2 min.

8. Place the column in a 1.5 mL sterilized centrifuge tube, add 30 μL
ddH2O to the center of the column, and leave for 2 min. Centrifuge at
12,000 rpm (13,800 × g) for 1 min. Store the DNA at -20 °C.

### PCR Solution Recovery Program

1. Briefly centrifuge PCR products. Measure the volume and transfer to a
1.5 mL centrifuge tube. If the sample volume is less than 100 μL,
replenish it to 100 μL with H2O.

2. Add 5 times the volume of Buffer GDP and mix by inversion or
vortexing.

3. Place the column in a collection tube. Transfer ≤700 μL of the solvent
to the column and centrifuge at 12,000 rpm (13,800 × g) for 60 sec.

(If the volume of lysate is >700 μL, return the column to the
Collection Tubes, and repeat step 3)4.

4. Follow steps 5 - 8 of Gel Recovery Program.

Supplement note

### 1. culture mediums(1000 mL)

LB liquid medium:

| Component | Amount |
| --- | --- |
| Yeast extract | 5g |
| tryptone | 10g |
| NaCl | 10g |

2x YT liquid medium:

| Component | Amount |
| --- | --- |
| Yeast extract | 10g |
| tryptone | 16g |
| NaCl | 5g |

BHI liquid medium:

| Component | Amount |
| --- | --- |
| Brain Heart Infusion Broth (Dehydrated) | 38.5g |

LB solid medium

| Component | Amount |
| --- | --- |
| Yeast extract | 5g |
| tryptone | 10g |
| NaCl | 10g |
| agar | 15g |

LB +v2 salts liquid medium:

| Component | Amount |
| --- | --- |
| Yeast extract | 5g |
| tryptone | 10g |
| NaCl | 129.34g (10g + 204mM×1L) |
| KCl | 3.12g (4.2mM × 1L) |
| MgCl2 | 24g（23.14mM × 1L） |
| K2HPO4 | 30.6g (17.6mM × 1L) |

BHI +v2 salts liquid medium:

| Component | Amount |
| --- | --- |
| Brain Heart Infusion Broth (Dehydrated) | 38.5g |
| NaCl | 119.34g (204mM×1L) |
| KCl | 3.12g (4.2mM × 1L) |
| MgCl2 | 24g（23.14mM × 1L） |
| K2HPO4 | 30.6g (17.6mM × 1L) |

### 2.other reagents used

Coomasie brilliant blue destaining solution

| Component | Volume |
| --- | --- |
| methanol | 250 mL |
| glacial acetic acid | 80 mL |
| ddH2O | To 1000 mL |

transformation storage buffer(a modified version of the buffer of
Inoue34 containing DMSO)

55 mM MnCl2, 15 mM CaCl2, 250 mM KCl, 10 mM PIPES (from 0.5 M, pH 6.7,
stock), 7% (v/v) spec grade DMSO (where the DMSO is added after cells
are suspended in the other buffer components)

| Component | Concentration |
| --- | --- |
| MnCl2 | 55 mM |
| CaCl2 | 15 mM |
| KCl | 250 mM |
| PIPES | 10 mM (from 0.5 M, pH 6.7, stock) |
| DMSO (added after cells are suspended in the other buffer components) | 7% (v/v) |

### 3.antibiotic concentration

Ampicillin storage solution:100 mg/mL

Kanamycin storage solution: 100 mg/mL

Ampicillin for single resistance Vibrio natriegens liquid culture: 100
μg/μL

Kanamycin for single resistance Vibrio natriegens liquid culture: 100
μg/μL

Double resistance Vibrio natriegens liquid culture: Ampicillin 75
μg/μL;Knamycin 75 μg/μL

Ampicillin for single resistance E.coli liquid culture: 100 μg/μL

Kanamycin for single resistance E.coli liquid culture: 50 μg/μL

Ampicillin for LB solid medium(E.coli):50 μg/μL

Ampicillin for LB solid medium(V.natriegens):30 μg/μL

### 4.primers

| name | function | sequence |
| --- | --- | --- |
| PLF | Screening of transformants of pPilin by colony PCR | GTCTGAGCGGTTATCAGAGACAACAG |
| PLR | Screening of transformants of pPilin by colony PCR | ATTGTCCATATTGCATCAGACATTGCC |
| CRF | Screening of transformants of pdCas9-sg1 by colony PCR | GGCACAAATAGCGTCGGATGG |
| CRR | Screening of transformants of pdCas9-sg1 by colony PCR | GCATCTACTCCACTTGCGTTAATAGGG |
| GFF | Screening of transformants of pGFP by colony PCR | GCACAAGCTGGAGTACAACTTCAAC |
| GFR | Screening of transformants of pGFP by colony PCR | ATTTGTCCTACTCAGGAGAGCGTTC |
| CutF | pGFP modification(reverse PCR,deleting the pilin fragment from the N terminus of sfGFP) | CATGAGAAGGaagagagaGAAATCAatggtgagcaagggc |
| CutR | pGFP modification(reverse PCR,deleting the pilin fragment from the N terminus of sfGFP) | ctctcttCCTTCTCATGAGATAGATAATGC |
| ModF1 | pGFP modification(reverse PCR,changing the position of initiation codon) | GAGAAGGaagagagaACGAAGTCAAATAAAC |
| ModR1 | pGFP modification(reverse PCR,changing the position of initiation codon) | CTTCGTtctctcttCCTTCTCATGAGATAG |
| ModF2 | pGFP modification(reverse PCR,changing the position of initiation codon) | GAATTAACGATCGTGGTGGCGGTAAT |
| ModR2 | pGFP modification(reverse PCR,changing the position of initiation codon) | CCACCACGATCGTTAATTCAATCAGC |

### 5.plasmids

| plasmid | backbone | resistance | Function gene | promoter | RBS | terminator |
| --- | --- | --- | --- | --- | --- | --- |
| pPilin | pUC19 | Ampicillin | Recombined Pil\_A | BBa\_I0500 | BBa\_B0034 | BBa\_B1006 |
| pGFP | pUC19 | Ampicillin | Recombined sfGFP | V.natriegens promoter for pilA | V.natriegensRBS for pilA | BBa\_B0010 |
| pdCas9-sg1 | pRSFDuet-1 | Kanamycin | sgRNA;spdCas9 | BBa\_J23100(sgRNA); BBa\_R0010(spdCas9) | BBa\_B0034(spdCas9) | BBa\_B1006 |

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

Sorry, your browser does not support inline SVG.
![logo](https://static.igem.wiki/teams/4786/wiki/img/sidenav-logo.svg)

## Results

[Wet lab result](#02)
[Strain construction](#03)
[Vibrio natriegens pili suppression](#04)
[e-Pili production](#05)
[Model result](#06)
[Optimization Results](#07)
[Model Evaluation](#08)
[Hardware result](#09)
[E-Pili Cartridge](#10)
[Plinker](#11)
[Back to top ⬆](#overview)

![overview_bg](https://static.igem.wiki/teams/4786/wiki/img/myoverview.png)

# Results

![](https://static.igem.wiki/teams/4786/wiki/img/overview.png)

## Introduction

This year, we aim to express these e-pili in Vibrio natriegens and establish a quantitative
antibody detection system based on this material. (To learn more:  [Design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D)).On this page, we will introduce the
results we have achieved during this project cycle.

今年，我们的目标是在需钠弧菌中表达这些电子毛发，并建立基于这种材料的定量抗体检测系统（详见 [Design](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27design%27%29%20%7D%7D)界面）。在这个页面上，我们将介绍我们在这个项目周期内取得的成果。

## Wet lab result

## Strain construction

There are two main strains that we constructed in our project. One of them is the pilin
producing strain V.PC and the other one is the report strain V.CR-mod(fig.1 and fig.2). During the
strain construction process, we tried three different transformation protocols and finally found out the
method that works to our V.natriegens strain.

在这次的项目中，我们构建了两个主要的菌株。其中一个是生产导电菌毛的V.PC菌株，另一个是报告菌株V.CR-mod。在菌株构建的过程中，我们一共尝试了三种不同的转化方法，最终找到了有效的电转细胞制备方式与电转参数。

Strain V.PC has plasmid pPilin and pdCas9-sg.pPilin(fig.1a)encodes e-pili monomer gene from
Geobactor metallireducens, designated as G.PilA. pdCas9-sg(fig.1b) encodes dCas9 from Streptococcus
pyogenes and sgRNA that targets at the promoter of type Ⅳ pilus monomer in V.natriegens(V.PilA
promoter). V.CR-mod is an accessory strain which also has two plasmids, pGFPmod2 and pdCas9-sg.
pGFPmod2(fig.2a) is a report plasmid to test the efficiency of CRISPRi system. sfGFP is under the
control of V.PilA promoter, which is the target of sgRNA in pdCas9-sg.

菌株V.PC转入了pPilin和pdCas9-sg两个质粒。pPilin编码金属还原地杆菌导电菌毛的组装单体，被命名为G.PilA。pdCas9-sg编码来自化脓链球菌的dCas9基因以及靶向需钠弧菌四型菌毛单体启动子（V.PilA
promoter）的sgRNA。V.CR-mod是一个辅助菌株，它也就有两个质粒，分别是pGFPmod2和pdCas9-sg。pGFPmod2是一个用于测试CRISPRi系统工作效率的报告质粒。在这个报告质粒中，sfGFP的表达由V.PilA
promoter控制，而V.PilA promoter正是CRISRPi质粒pdCas9-sg中sgRNA的靶向位点。

![](https://static.igem.wiki/teams/4786/wiki/img/results/result1-1.png)
![](https://static.igem.wiki/teams/4786/wiki/img/results/results1-2.png)![](https://static.igem.wiki/teams/4786/wiki/img/results/results1-3.jpeg)

Fig 1 a)Structure of plasmids in V.PC b)The colony PCR result of
V.PC

图1 (a)V.PC中的质粒结构 (b)V.PC 菌落PCR结果

![](https://static.igem.wiki/teams/4786/wiki/img/results/result2-1.png)![](https://static.igem.wiki/teams/4786/wiki/img/results/results2-2.png)![](https://static.igem.wiki/teams/4786/wiki/img/results/results2-3.png)

Fig 2. a)Structure of plasmids in V.CR-mod b)The colony PCR result of
V.CR-mod

## Vibrio natriegens pili suppression

### CRISPRi system construction

Because Vibrio natriegens itself can produce type Ⅳ pilus, we need to suppress the endogenous
production of type Ⅳ pilus before producing the e-pili. Therefore, we designed a CRISPRi system to
suppress the expression of V.PilA.The reason why we didn't choose CRISPR to knock out V.PilA was
that it has been reported in the literature that DNA double-stranded breaks caused by the
CRISPR/Cas9 system could be extremely toxic to Vibrio spp because non-homologous end-joining (NHEJ)
activity is almost undetectable in the microorganism.

因为需钠弧菌本身可以产生Ⅳ型鞭毛，所以在产生e-鞭毛之前，我们需要抑制内源性Ⅳ型鞭毛的产生。因此，我们设计了一个CRISPRi系统来抑制V.PilA的表达。我们之所以没有选择CRISPR来敲除V.PilA，是因为文献中报道了CRISPR/Cas9系统引起的DNA双链断裂对嗜盐弧菌等微生物的毒性极高，因为非同源末端连接（NHEJ）活性在该微生物中几乎无法检测到。

We initially designed four different sgRNAs targeting at the sequence near transcription start
site(TSS) of V.PilA. We ordered the first version of pdCas9-sg which had sgRNA 1 and gene segments
containing the other three sgRNAs. Then, using high fidelity reversed PCR and Gibson assembly, we
acquired 4 different versions of pdCas9-sg plasmids(fig.3b).

我们最初设计了四种不同的 sgRNA，靶向 V.PilA 的转录起始位点（TSS）附近的序列。我们订购了第一版 pdCas9-sg，其中包含 sgRNA 1 和含有其他三个
sgRNA 的基因片段。然后，通过高保真反向 PCR 和 Gibson 组装，我们获得了 4 个不同版本的 pdCas9-sg 质粒（图 3b）。

![](https://static.igem.wiki/teams/4786/wiki/img/results/results3-1.png)
![](https://static.igem.wiki/teams/4786/wiki/img/results/results3-2.png)
![](https://static.igem.wiki/teams/4786/wiki/img/results/results3-3.png)

Fig 3 a) CRISPRi efficiency measurement b)use Gibson assembly to acquire
different CRISPRi plasmids c) CRISPRi targets.

### System efficiency measurement

pGFPmod2 is the final version of our report plasmid for CRISPRi efficiency measurement.(for
designing process of report plasmid, visit [engineering success](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D) ) It has
the gene
of sfGFP under the control of V.PilA promoter and the N terminus sequence of V.PilA links to the N
terminus of sfGFP. The methionines in the V.PilA sequence was mutated so the folding of sfGFP would
not be influenced by an attached fragment(fig.4).

pGFPmod2是我们用于CRISPRi效率测定的报告质粒的最终版本（报告质粒的设计过程请访问[engineering
success](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D)页面），它具有V.PilA启动子控制的sfGFP基因，V.PilA的N末端序列与sfGFP的N末端相连。V.PilA 序列中的蛋氨酸被突变，因此 sfGFP
的折叠不会受到连接片段的影响（图
4）。

![](https://static.igem.wiki/teams/4786/wiki/img/results/results4-1.png)

Fig 4 construction of pGFPmod2

![](https://static.igem.wiki/teams/4786/wiki/img/results/results4-2.png)

Fig 5 Fluorescence intensity measurement of two versions of report
plasmid

pGFPmod2 was cotransferred with pdCas-sg,pdCas-sg2,pdCas-sg3 and pdCas-sg4 seperatly into
V.natriegens(fig .6). Overnight cultures of these four strains were spread out on plates and the cells
were collected with culture medium for Green flourescence measurement. Based on the measurement results,
it can be observed that all CRISPRi plasmids, except for pdCas9-sg1, have a certain inhibitory effect.
Among them, pdCas9-sg4 shows the best inhibitory effect. However, due to time constraints, we only used
the initial pdCas9-sg1 plasmid to construct the bacterial strain for pilus expression.

将 pGFPmod2 与 pdCas-sg、pdCas-sg2、pdCas-sg3 和 pdCas-sg4 分别共转到需钠弧菌菌株中（图
6）。这四个菌株的过夜培养物被涂布在平板上，并用培养基收集细胞进行绿色荧光测量。从测量结果可以看出，除了pdCas9-sg1之外，其余CRISPRi质粒均有一定抑制效果，其中pdCas9-sg4的抑制效果最佳。但由于时间限制，我们仅使用了最初的pdCas9sg-1质粒进行菌毛表达菌株的构建。

![](https://static.igem.wiki/teams/4786/wiki/img/results/fig6.png)

Fig 6 Suppression efficiency measurement of four CRISPRi plasmids

图6 四种不同CRISPRi质粒抑制效率的测量

## e-Pili production

### Pili harvest

Overnight culture of e-Pili producing strain V.PC was spread out on plates for pili harvest.
Meanwhile,
we also spread overnight wild type V.natriegens culture on plates.It could be seen that the apprearance
of lawn of these two strains were different.The lawn of V.PC, which produced e-Pili of
G.metallireducens, had winkled surface(fig.7a), while the lawn of wild type V.natriegens was
smooth(fig.7b).

将产生导电菌毛的菌株 V.PC 的过夜培养物铺展在平板上进行导电菌毛收获。从图 7a 可以看出，产生金属还原地杆菌导电菌毛的 V.PC 菌株的平板表面呈褶皱状，而野生型
需钠弧菌 菌株的平板表面则很光滑（图 7b）

![](https://static.igem.wiki/teams/4786/wiki/img/results/results5-1.png)![](https://static.igem.wiki/teams/4786/wiki/img/results/results5-2.png)

Fig 7 Different lawn appearance of a)V.PC and b)wild type V.natriegens.

The whole procedure of e-pili harvest included cell collection and resuspension, shearing pili
from
cells, removal of cell debris and precipitation of e-pili.The harvested e-pili solution was on average
2~3 μg/μL.

收获导电菌毛的整个过程包括细胞收集和重悬浮、剪切细胞中的导电菌毛、清除细胞碎片和沉淀导电菌毛。

The harvested pili solution was purified using Ni-NTA resin. To roughly test the conductivity
of this
pili, we used gold electrode and multimeter to measure the resistance of the unpurified e-pili solution
and purified solution. Pili solution was dropped between two gold electrodes and waited about 10 min for
the solution to air-dry. After air-drying, the pili solution became a protein film. Resistance
was measured when the protein film was still moisted. The vedios below illustrate different resistance
of purified or unpurified e-pili solution. It could be seen that the resistance of unpurified pili
solution had a resistance of about 24.3kΩ while purified pili solution had a lower resistance of about
17kΩ.

We also measured the resistance of ddH2O we used to solve the pili and NaCl solution(5%) as
control.The resistance of ddH2O was about 1.87MΩ and that of the NaCl solution was about 57 kΩ.We also
performed the same test on BSA standard solution(adjusted to the same concentration as purified pili
solution) and the result was about 220 kΩ.

为了粗略测试导电菌毛的电导率，我们使用金电极和万用表测量了未经纯化的导电菌毛溶液和纯化溶液的电阻。将导电菌毛溶液滴在两个金电极之间，等待约 10 分钟使溶液风干。风干 20
分钟后，导电菌毛溶液变成一层蛋白质膜。下面的图片展示了纯化和未纯化导电菌毛溶液的不同电阻。可以看出，未经纯化的导电菌毛溶液的电阻约为 24.3kΩ，而纯化的导电菌毛溶液的电阻较低，约为 17kΩ。

Unpurified e-pili solution

purified e-pili solution

ddH2O

10%Nacl

BSA standard

![](https://static.igem.wiki/teams/4786/wiki/img/results/output.png)

Fig 8 Conductance of different solutions

Then, we used TEM (transmission electron microscope) to view the micro-structure of the
pilis. In fig.9, several nanowires could be seen after negative staining (fig.9) However, there were
many impurities in our sample as shown by the TEM result. We speculated that these impurities may be
caused by insufficient speed provided by our centrifuge and unskilled operation of purification using
Ni-NTA resin. Apart from that, there were much fewer pili in our sample compared with samples harvested
in previous research[1]. We inferred that the expressing efficiency or assembly ability of G.PilA in
V.natriegens was not enough. It could be part of the reason why we failed to detect G.PilA in Western
Blot that the concentration of His-tagged pili was lower than the detection threshold.

然后，我们使用 TEM（透射电子显微镜）观察菌毛的微观结构。从图 6 中可以看到，经过负染色后，可以看到几条纳米线（图 6）。然而，从 TEM
的结果来看，我们的样品中有很多杂质。我们推测，这些杂质可能是由于我们的离心机速度不够快，以及使用 Ni-NTA
树脂纯化时操作不熟练造成的。此外，与之前的研究[1]相比，我们的样本中的菌毛数量要少得多。我们推断，G.PilA 在 需钠弧菌 中的表达效率或组装能力不够。这可能是我们未能在 Western
blotting中检测到 G.PilA 的部分原因，因为 His 标记的菌毛浓度低于检测阈值。

![](https://static.igem.wiki/teams/4786/wiki/img/results/results7-1.jpg)

Fig 9 (a)The result of TEM. Nanowires were marked with white arrows.

![](https://static.igem.wiki/teams/4786/wiki/img/results/results7-2.png)

Fig 9 (b) TEM of pili of Geobacter metallireducens from previous
reseach[1].

To validate the concept of using conductive pili to measure concentrations of analytes in
samples, we
conducted preliminary tests using harvested and purified e-pili solutions. While maintaining a
relatively constant filament concentration, we introduced equal concentrations of different
antibodies (with antibody solution volume much smaller than the filament solution volume) into the
solution. The anti-His tag antibody was able to bind to the fusion His-tagged filaments, while the
anti-GAPDH antibody, an irrelevant antibody, served as a control. The results of the test are shown
in Figure 10. It can be observed that the addition of anti-His tag antibody significantly increased
the resistance of the solution, while the addition of irrelevant anti-GAPDH antibody resulted in a
smaller change in resistance. Although this test was relatively crude and the results are not highly
significant, it provided a positive foundation for further improvement in our project. Based on this
foundation, we can optimize the theoretical framework, experimental design, and operation to get
closer to our predetermined goals.

为了检验用导电菌毛来测量样品中待测物浓度的设想，我们用收获并纯化的菌毛溶液进行了初步的测试。在保证菌毛浓度基本不变的前提下，我们向其中加入了相同浓度的不同抗体（抗体溶液体积远小于菌毛溶液体积），其中抗His
tag抗体可以与融合His tag的菌毛相结合，而抗GAPDH抗体为无关抗体作为对照。测试结果如图10所示。可以看出，在加入抗His
tag抗体后溶液的电阻有较大的提高，而加入无关的抗GAPDH抗体后阻值变化则较小。虽然这一测试较为粗糙，且结果不是十分显著，但这为我们项目后续进一步完善提供了较为积极的的基础，在该基础上进行理论、实验设计、操作等的优化，希望能够进一步向我们预设的目标靠近。

![](https://static.igem.wiki/teams/4786/wiki/img/results/f10n.png)

Fig 10 Influence of antibody of the conductance of pili solution.

## Model result

## Optimization Results

We can observe that adding new aromatic amino acids can significantly optimize both our
objective functions one and two.

可以看到，添加新的芳香族氨基酸可以显著优化目标函数一和目标函数二。

![](https://static.igem.wiki/teams/4786/wiki/img/results/results8-1.png)![](https://static.igem.wiki/teams/4786/wiki/img/results/results8-2.png)

Fig.11 Optimization Resultss

**In objective function one** , we found that replacing serine with aromatic
amino acids at position 2 of the original amino acid sequence of the e-pili can reduce the average
distance in objective one from 4.72 Å to 4.63Å. To verify the accuracy of the results, we attempted to
iterate through all possible spacing scenarios for adding 1, 2, and 3 pi-residues (Fig. 2) and found
that only the modification at position 2 changes the original shortest electron transfer pathway, and
adding more aromatic amino acids does not alter this result.（Fig.9）

在目标函数一中，我们发现在 e-pili 原始氨基酸序列的第 2 位用芳香族氨基酸替换丝氨酸可以将目标函数一中的平均距离从 4.72 Å 缩小到 4.63 Å。
为了验证结果的准确性，我们尝试遍历了添加 1、2 和 3 个 pi残基的所有可能的间距方案（图 2），结果发现只有第 2
位的修饰改变了原来的最短电子传递途径，而添加更多的芳香族氨基酸并不会改变这一结果。（图9）

![](https://static.igem.wiki/teams/4786/wiki/img/results/results9.png)

Fig.12 Results of traversing with the addition of 1, 2, and 3
Π-residues.

**In objective function two** , because the objective function we chose is used
to describe the distribution density of aromatic rings within the e-pili, this means that adding 1 or 2
pi-residues cannot yield an optimal result. Therefore, we started considering the addition of three
Π-residues, as shown in Fig. 1. Adding 5 aromatic amino acids can reduce the original spacing from 7.6 Å
to less than 5.5 Å.

在目标函数二中，由于我们选择的目标函数用于描述 e-pili 中芳香环的分布密度，这意味着添加 1 个或 2 个
pi残基无法获得最佳结果。因此，我们开始考虑添加三个Π-残基，如图 1 所示。添加 5 个芳香族氨基酸可以将原来的间距从 7.6 Å 缩小到 5.5 Å 以下。

## Model Evaluation

Based on the optimization results from the model described above, we reconstructed the
structural models for single point mutants T2F and triple point mutants A31F, Q53F, and N59F to validate
our conclusions:

根据上述模型的优化结果，我们重建了单点突变体 T2F 和三点突变体 A31F、Q53F 和 N59F 的结构模型，以验证我们的结论：

**In optimization objective one** , we replaced the threonine at position 2 of
the wild-type metal-reducing rod bacterium pilin amino acid sequence with phenylalanine. We only
selected the shortest electron transfer path in the pilus structure obtained by the Dijkstra algorithm
(where the original aromatic rings are marked in red, and the newly added aromatic rings in mutant T2F
are marked in yellow).

在优化目标一中，我们用苯丙氨酸替换了野生型金属还原地杆菌菌毛氨基酸序列中第 2 位的苏氨酸。我们只选择了通过 Dijkstra
算法得到的柔毛菌结构中最短的电子传递路径（原芳香环用红色标出，突变体 T2F 中新添加的芳香环用黄色标出）。

![](https://static.igem.wiki/teams/4786/wiki/img/results/results10.png)

Fig 13 The electron transport chain in Mutant T2F

We found that the newly added aromatic amino acids changed the original electron transfer
chain to (F1p, Y27p+4, F24p+4) (electrons are transferred from position 1 of the first monomer to
positions 27 and 24 of the fifth monomer, and finally to position 1 of the second monomer to complete
the cycle), where the centroid distances between adjacent aromatic rings are (4.6 Å, 5.5 Å, 4.1 Å). In
the mutant, according to our model prediction, the electron transfer chain would change to (F2p, Y27p+4,
F24p+4). However, we can clearly observe that the centroid distance between the aromatic rings in the
electron transfer chain of this mutant is significantly larger than that of the wild type, with
distances of (7.1 Å, 5.5 Å, 9.5 Å). （Fig11）

我们发现，新添加的芳香族氨基酸将原来的电子传递链改变为（F1p, Y27p+4, F24p+4）（电子从第一个单体的第 1 位转移到第五个单体的第 27 位和第 24
位，最后转移到第二个单体的第 1 位，完成循环），其中相邻芳香环之间的中心距为（4.6 Å, 5.5 Å, 4.1
Å）。在突变体中，根据我们的模型预测，电子传递链将变为（F2p、Y27p+4、F24p+4）。然而，我们可以清楚地观察到，该突变体电子传递链中芳香环之间的中心距明显大于野生型，分别为（7.1 Å, 5.5
Å, 9.5 Å）。（图11）

![](https://static.igem.wiki/teams/4786/wiki/img/results/results11.png)

Fig14.The comparison in electron transport chain between the
WT（yellow）and the T2F（orange）

This situation arose because, when determining the constraint conditions of the decision
variables in the optimization model, to simplify the model, we used the average coordinates of all
carbon atoms on the side chain of the modified position to approximately predict the centroid
coordinates of the newly added aromatic ring. The error caused by simplifying the model ultimately led
to incorrect optimization results.

This model illustrates that objective function one can no longer be optimized by adding
new aromatic amino acids to the original amino acid sequence. Subsequent teams may try to optimize by
changing the type of aromatic amino acids to affect the overall hydrophilicity and hydrophobicity of the
pilus.

This model illustrates that objective function one can no longer be optimized by adding new
aromatic amino acids to the original amino acid sequence. Subsequent teams may try to optimize by
changing the type of aromatic amino acids to affect the overall hydrophilicity and hydrophobicity of the
pilus.

该模型说明，在原始氨基酸序列中添加新的芳香族氨基酸已无法优化目标函数一。后续团队可能会尝试通过改变芳香族氨基酸的类型来优化，从而影响菌毛的整体亲水性和疏水性。

**In objective function two**, we added 3, 4, and 5 phenylalanine residues at the
positions calculated by our optimization algorithm, respectively, on top of the original structure. We
can intuitively observe that the introduction of aromatic amino acids significantly improves the uniform
distribution of aromatic amino acids in the original pili structure, causing the core of electron
transfer to spread outward from the pili's central axis. This means that when the structure of
conductive pili changes under the influence of different environmental factors, there are more aromatic
rings involved in the electron transfer process, potentially leading to an increase in the overall
electrical conductivity of the pili.

在目标函数二中，我们在原始结构的基础上，在优化算法计算出的位置上分别添加了 3、4 和 5
个苯丙氨酸残基。我们可以直观地观察到，芳香族氨基酸的引入明显改善了原始菌毛结构中芳香族氨基酸的均匀分布，使电子传递的核心从菌毛的中轴向外扩散。这意味着当导电菌毛的结构在不同环境因素的影响下发生变化时，有更多的芳香环参与电子传递过程，从而有可能导致菌毛整体导电率的提高。

![](https://static.igem.wiki/teams/4786/wiki/img/results/results12.jpg)

Fig15. Optimization results of objective function 2

## Hardware result

In our design, since our system needs to detect samples with possible pathogenic hazards. In
order to maximize the protection of the operator of this hardware, we broke down our hardware into two
isolated parts, a test disposable and a multi-use measurement and data processing device.

在我们的设计中，由于我们的系统需要检测具有可能的病原危害性的样本。为了最大程度的保护这台硬件的操作者，我们将我们的硬件分解成一次性检测耗材和多次使用的测量和数据处理装置这两个之间相互隔离的部分。

For the hardware part we prototyped the quantitative rapid test device Plinker and the test
disposable E-Pili Cartridge. We designed the PCB for the test module and debugged and packaged the WiFi
module and screen. We achieved successful connectivity between the two parts by designing and iterating
on the connection method, and the Plinker was able to measure the conductivity of the E-Pili in the
Cartridge.

硬件部分我们制作了定量快速检测设备Plinker和检测耗材E-Pili
Cartridge的原型。我们设计了检测模块的PCB，并进行了WiFi模块、屏幕的调试和封装。我们通过设计和迭代连接方式，实现了两部分之间成功的连通，Plinker可以测量出Cartridge中菌毛的电导率。

To demonstrate the relative accuracy of our system, we also designed and did a simulation
experiment to test the results of Plinker's conductivity measurements using a saline solution and a
buffer. The results did prove the accuracy of our system.

为了证明我们的系统相对精确，我们还设计并进行了一个模拟实验，使用盐溶液和缓冲液测试Plinker测量电导率的结果。结果的确证明了我们系统的精确性。

## E-Pili Cartridge

The E-Pili Cartridge needed to provide a platform that would allow full binding of the
modified E-Pili and the disease marker to be tested. After several iterations we finally settled on
using lateral flow immunochromatography, the most common structure used in rapid screening kits, as the
substrate for our disposables. We immobilize the purified E-Pili in the test region on the NC membrane,
and the sample solution added on the other side will flow across the NC membrane under the moisture
gradient provided by the absorbent pad, and eventually bind to the E-Pili in the test region. We placed
a interdigital electrode in the upper layer of the E-Pili, customized a new casing to accommodate the
electrode, and designed its connection to the Plinker for measurement purposes.

E-Pili
Cartridge需要提供一个让修饰后的菌毛和待测疾病标志物充分结合的平台。经过了多次的迭代后我们最终确定了使用快速筛查试剂盒最常用的结构---侧流免疫层析作为我们的耗材的基底。我们将纯化后的导电菌毛固定在NC膜上的待测区域中，在另一侧加入的待测样品溶液会在吸水垫提供的水分梯度的作用下在NC膜上流动，最终在待测区域和菌毛结合。我们在菌毛上层置入叉指电极，定做了新的可以容纳电极的外壳，并且设计了其与Plinker的连接方式以达成测量的目的。

![](https://static.igem.wiki/teams/4786/wiki/img/results/design13.png)

Fig.16 E-Pili Cartridge Prototype\_V2

## Plinker

The accuracy of quantification is directly proportional to the accuracy of the conductivity
measuring instrument. In order to get better quantitative results, we designed a high accuracy
conductivity measurement system based on the STC15 development board. We made it as a PCB for detecting
the resistance value and analyzing it.

定量的准确程度和电导率测量仪器的精度成正比。为了获得更加好的定量结果，我们设计了一块基于STC15开发板的高精度电导率测量系统。我们将其制作为PCB，用于检测电阻值和分析。

In addition, the conductivity of the E-Pili may be affected by the ambient temperature and
humidity. As a system with high requirements for quantitative accuracy, our hardware must be able to
realize the quality control of the data it produces and the automatic correction within a certain error
range. For this purpose, we have designed a Blank program and a quality control step in this module.

除此之外，菌毛的电导率可能会受到环境温度和湿度的影响，作为一个对定量的精度具有较高要求的系统，我们的硬件必须要能够实现对于其所产生的数据的质量控制和在一定误差范围内的自动修正。为此我们在此模块中设计还设计了Blank程序和质检步骤。

Finally, we used 3D printing and laser cutting to create a casing for the Plinker that
seamlessly connects to the Cartridge, and embedded electrodes to connect the two parts.

最后我们用3D打印和激光切割技术为Plinker制作了一个可以与Cartridge无缝连接的外壳，并在其中嵌入用于连接的电极，以连通这两部分。

![](https://static.igem.wiki/teams/4786/wiki/img/results/design14.png)

Fig.17 Before and after Inserting E-Pili Cartridge into Plinker

### WiFI module

We download a TCP client software on the mobile device and input the IP address and port number of the
ESP8266 module for connection testing. This is done to ensure that communication is working correctly.
This process verifies the stability of data transmission and reliability of communication for the WiFi
module.

![](https://static.igem.wiki/teams/4786/wiki/img/results/f18.png)

Fig。19 Simulation experiment result

### Simulation Experiment

We added Tris-HCl buffer to the sample hole and again added Tris-HCl buffer with an additional 5% KCL at the sample hole when the conductivity indication stabilized. The conductivities of these two solutions were measured individually (by adding these two solutions dropwise directly to the interdigital electrode) as 3.134 mS and 3.845 mS, respectively. Finally, we can get the following results:

![](https://static.igem.wiki/teams/4786/wiki/img/results/f19.png)

Fig 19 Simulation experiment result

We can observe that after the addition of the Tris-HCl buffer, the buffer flowed laterally across the NC membrane, and eventually the number stabilized at 2.712 mS (after about 1 minute). After the number stabilized, we added Tris-HCl buffer with an additional 5 percent KCl, and the number started to change after about 15 seconds, and finally the number stabilized at 3.533 mS (about 1 minute).

Although there is a certain error (0.5mS) between the conductivity value measured after chromatography and that measured by directly adding the solution dropwise to the interdigital electrode, the difference between the conductivities of different ion concentrations measured by the two methods is relatively close (0.711mS vs. 0.821mS). This result can be good proof that our system is relatively accurate.

### Proof of concept

We tested the conductance of pilin solution after adding different and concentration of anti-His antibody. As shown in this figure, when adding serial concentrations of appropriate antibody into the e-pili solution, the resistance elevated gradually between zero and 150 micrograms per mililiter, then the curve reached a plateau after that. In contrast, pili solution of wild type Vibro natriegens harvested with the same method didn't show this characteristic.

![](https://static.igem.wiki/teams/4786/wiki/img/results/fend1.png)
![](https://static.igem.wiki/teams/4786/wiki/img/results/fend2.png)

{% endblock %}