iGEM 2022 — Team nycu-formosa

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block page\_content %}

Lab notes

Wet Lab

Dry Lab

Human Practice

* ❮
* ❯
* June
  2022

* Sunday
* Monday
* Tuesday
* Wednesday
* Thursday
* Friday
* Saturday

* + 29
  + 30
  + 31
  + 1
  + 2
  + 3
  + 4
* + 5
  + 6
  + 7
  + 8
  + 9
  + 10
  + 11
* + 12
  + 13
  + 14
  + 15
  + 16
  + 17
  + 18
* + 19
  + 20
  + 21
  + 22
  + 23
  + 24
  + 25
* + 26
  + 27
  + 28
  + 29
  + 30
  + 1
  + 2
* + 3
  + 4
  + 5
  + 6
  + 7
  + 8
  + 9

* ❮
* ❯
* July
  2022

* Sunday
* Monday
* Tuesday
* Wednesday
* Thursday
* Friday
* Saturday

* + 26
  + 27
  + 28
  + 29
  + 30
  + 1
  + 2
* + 3
  + 4
  + 5
  + 6
  + 7
  + 8
  + 9
* + 10
  + 11
  + 12
  + 13
  + 14
  + 15
  + 16
* + 17
  + 18
  + 19
  + 20
  + 21
  + 22
  + 23
* + 24
  + 25
  + 26
  + 27
  + 28
  + 29
  + 30
* + 31
  + 1
  + 2
  + 3
  + 4
  + 5
  + 6

* ❮
* ❯
* August
  2022

* Sunday
* Monday
* Tuesday
* Wednesday
* Thursday
* Friday
* Saturday

* + 31
  + 1
  + 2
  + 3
  + 4
  + 5
  + 6
* + 7
  + 8
  + 9
  + 10
  + 11
  + 12
  + 13
* + 14
  + 15
  + 16
  + 17
  + 18
  + 19
  + 20
* + 21
  + 22
  + 23
  + 24
  + 25
  + 26
  + 27
* + 28
  + 29
  + 30
  + 31
  + 1
  + 2
  + 3
* + 4
  + 5
  + 6
  + 7
  + 8
  + 9
  + 10

* ❮
* ❯
* september
  2022

* Sunday
* Monday
* Tuesday
* Wednesday
* Thursday
* Friday
* Saturday

* + 28
  + 29
  + 30
  + 31
  + 1
  + 2
  + 3
* + 4
  + 5
  + 6
  + 7
  + 8
  + 9
  + 10
* + 11
  + 12
  + 13
  + 14
  + 15
  + 16
  + 17
* + 18
  + 19
  + 20
  + 21
  + 22
  + 23
  + 24
* + 25
  + 26
  + 27
  + 28
  + 29
  + 30
  + 1
* + 2
  + 3
  + 4
  + 5
  + 6
  + 7
  + 8

* ❮
* ❯
* October
  2022

* Sunday
* Monday
* Tuesday
* Wednesday
* Thursday
* Friday
* Saturday

* + 25
  + 26
  + 27
  + 28
  + 29
  + 30
  + 1
* + 2
  + 3
  + 4
  + 5
  + 6
  + 7
  + 8
* + 9
  + 10
  + 11
  + 12
  + 13
  + 14
  + 15
* + 16
  + 17
  + 18
  + 19
  + 20
  + 21
  + 22
* + 23
  + 24
  + 25
  + 26
  + 27
  + 28
  + 29
* + 30
  + 31
  + 1
  + 2
  + 3
  + 4
  + 5

✖

## 2022.06.23

### IDT gblock resuspend

A, B, C, D, E, F

### Taq PCR

A, B, C, D, E, F

### Gel electrophoresis

A, B, C, D, E, F

✖

## 2022.06.29

### Transformation

pSB1A3 in DH5α

✖

## 2022.06.30

### Colony PCR

Pick four strains of bacteria from plate: 1, 2, 3, 4

### Gel electrophoresis

1, 2, 3, 4

✖

## 2022.07.01

### Culturing

Pick two from each area: 1-1, 1-2, 2-1, 2-2, 3-1, 3-2, 4-1, 4-2

✖

## 2022.07.02

### Plasmid purification

1-1, 1-2, 2-1, 2-2, 3-1, 3-2, 4-1, 4-2

✖

## 2022.07.04

### Phusion PCR & Gel extraction

A, B

### Digestion

A, B

### Gel extraction

A, B

### Ligation

A, B

✖

## 2022.07.05

### Phusion PCR & Gel extraction

E, F

### Digestion

E, F

### Gel extraction

E, F

### Ligation

E, F

### Transformation

A, B

✖

## 2022.07.06

### Phusion PCR & Gel extraction

C, D

### Digestion

C, D

### Gel extraction

C, D

### Ligation

E, F

### Transformation

E, F

### Colony PCR & Gel electrophoresis

Pick eight plants from each plate A & B
A: three from each II, III, V area (A-2, A-3, A-5)
B: three from each IV, VIII (B-4, B-8)

✖

## 2022.07.07

### Transformation

E, F

### Colony PCR & Gel electrophoresis

E => Pick three plants from plate

### Ligation

E, F

### Transformation

C, D

### Culturing

A, B: Three tubes from each: A-2, A-3, A-5, B-4, B-8 (15 tubes in total)

✖

## 2022.07.08

* A: His Ubx
* B: His EGFP Ubx
* C: PaPilA Ubx
* D: PaPilA 1-61 Ubx
* E: His Y4 mRFP Y293Y296
* F: His Y167 mRFP Y240

### Colony PCR

E, F => Pick eight plants from each plate
E: E-1,E-2, E-3,E-4,E-5,E-6,E-7, E-8
F: F-1, F-2, F-3,F-4,F-5,F-6,F-7., F-8

### Gel electrophoresis

Eight plants from E, F plate

### Plasmid purification

Three tubes from each: A-2, A-3, A-5, B-4, B-8

### Ligation

C

✖

## 2022.07.18

### Transformation

C, D, E, F

✖

## 2022.07.19

### Colony PCR & Gel electrophoresis

Pick eight plates from each plate: C, D, E, F
C: C-1, C-2,C-3,C-4,C-5,C-6,C-7, C-8
D: D-1, D-2, D-3, D-4, D-5, D-6, D-7, D-8
E: E-1, E-2, E-3, E-4, E-5, E-6, E-7, E-8
F: F-1, F-2, F-3, F-4, F-5, F-6, F-7, F-8

### Culturing

Choose two tubes from each: C-2, C-3, C-7, C-8

### Transformation

pSB3K3 in DH5α

### Colony PCR & Gel electrophoresis

Pick eight plates from each plate: C, D, E, F
C: C-1, C-2,C-3,C-4,C-5,C-6,C-7, C-8
D: D-1, D-2, D-3, D-4, D-5, D-6, D-7, D-8
E: E-1, E-2, E-3, E-4, E-5, E-6, E-7, E-8
F: F-1, F-2, F-3, F-4, F-5, F-6, F-7, F-8

✖

## 2022.07.20

### Colony PCR & Gel electrophoresis

pSB3K3 in DH5α

### Plasmid purification

two tubes from each: C-2, C-3, C-7, C-8

### Digest check

C-2, C-3, C-7, C-8

### Gel electrophoresis

Wrong result from digest check, so we use:
7/6 Digestion products
7/6 7/8 Ligation products

### Culturing

4 mL from each: pSB3K3 B, C, D

Two tubes of 3 mL from each:
pSB1A3 C-11, C-14, D-11, D-12, D-15, E-10, E-14, F-9, F-10, F-11, F-13, F-16

✖

## 2022.07.21

### Plasmid purification

C-11, C-14, D-11, D-12, D-15, E-10, E-14, F-9, F-10, F-11, F-13, F-16

pSB3K3-1, pSB3K3-2

### Digest check

C-11, C-14, D-11, D-12, D-15, E-10, E-14, F-9, F-10, F-11, F-13, F-16

### Digestion & Gel extraction

A-2, B-8, pSB3K3-1

### Transformation

pSB3K3 in DH5α

✖

## 2022.07.22

### Transformation

pSB1A3 A-2, B-8, C-14, D-11, E-14, F-11

### Colony PCR

pSB3K3 1~8, C-17, C-18, C-19, C-20

✖

## 2022.07.23

### Culturing

pSB3K3 1~8, C-17, C-18, C-19

### Plasmid purification

pSB3K3 1~8, C-17, C-18, C-19

### Digestion & Gel extraction

Backbone: pSB3K3 1

Insert: C-14, D-11, E-14, F-11

### Culturing

Take eight plants from pSB1A3
(C-14-1, C-14-2, D-11-1, D-11-2, E-14-1, E-14-2, F-11-1, F-11-2)

✖

## 2022.07.24

### Colony PCR

Take eight from each:
A-2, B-8, C-14, D-11, E-14, F-11 (pSB1A3 BL21)

### Plasmid purification

C-14, D-11, E-14, F-11 (pSB1A3)

### Digestion & Gel extraction

Backbone: pSB3K3 6
Insert: C-14, D-11, E-14, F-11

### Buffer W2 Testing

### Plasmid culturing

pSB1A3 BL21 A-2-2, A-2-5, B-8-2, B-8-5, C-14-7, C-14-8, D-11-1, D-11-8, E-14-1, E-14-7, F-11-3, F-11-7

✖

## 2022.07.25

### Digestion & Gel extraction

pSB3K3-1, pSB3K3-2, C-14, D-14, E-14, F-11

### Digest check

C-17, C-18, C-19 (DH5α)
A-2-2, A-2-5, B-8-2, B-8-5, C-14-7, C-14-8, D-11-1, D-11-8, E-14-1, E-14-7, F-11-3, F-11-7 (BL21)

### Ligation

A-2, B-8 (pSB3K3)

✖

## 2022.07.26

### Transformation

A-2, B-8 (pSB3K3 in BL21)

### IDT gblock resuspend

G: LL37 Ubx mRFP

### Taq PCR & Gel electrophoresis

G

### Phusion PCR & Gel extraction

G

✖

## 2022.07.27

### Colony PCR

A-2, B-8, C-14, D-11, E-14, F-11 (pSB1A3)

### Phusion PCR & Gel extraction

G

### Digestion

G

### Ligation

G in pSB1A3

✖

## 2022.07.28

### Transformation

pSB1A3 in BL21

### Plasmid purification

A-2-9, A-2-10

### Colony PCR

Three from each: A-2, B-8, C-14, D-11, E-14, F-11 (pSB1A3)

### Ligation

For start unit: pSB1A3-1, A-2-9-1, A-2-9-2
(Label: E1-C, E1-A-2-1, E1-A-2-2)

✖

## 2022.07.29

### Expression

E1-C, E1-A-2-1, E1-A-2-2

### Digest check

E1-A-2-1, E1-A-2-2

### Transformation

pSB1A3 in DH5α

### Plasmid purification

E1-A-2-1, E1-A-2-2

✖

## 2022.07.30

### Colony PCR

G (pSB1A3 DH5α)

✖

## 2022.08.01

### PCR for primer check

B: His EGFP Ubx

### Wash bacterial cells

Bacteria collected every hour from E1-A-2-1, E1-A-2-2

### Ligation

C(pSB1A3)

✖

## 2022.08.02

### Transformation

C, G (pSB1A3 in DH5α)

### Sonicate

Bacteria collected every hour from E1-A-2-1, E1-A-2-2

### Colony PCR

C-1, C-2, C-3 (pSB1A3-1 BL21)

For start unit: pSB1A3-1-1, D-11-9-5, D-11-9-6, F-11-10-4, F-11-10-5 (Label: E2-C, E2-D-1, E2-D-2, E2-F-1, E2-F-2)

✖

## 2022.08.03

### Expression

E2-C, E2-D-1, E2-D-2, E2-F-1, E2-F-2

### Plasmid purification

E2-D-1, E2-D-2, E2-F-1, E2-F-2

### Colony PCR

Four plants from each C, G (pSB1A3 DH5α)

For start unit: D

Four plants from each B, E

### Digest check

E2-D-1, E2-D-2, E2-F-1, E2-F-2

✖

## 2022.08.04

### Plasmid purification

C-2, C-4, G-3

### Colony PCR

C-1, C-2, C-3 (pSB1A3 BL21)

### Digest check

C-2, C-4, G-3

✖

## 2022.08.05

### Digest check

C-2, C-4, G-3

✖

## 2022.08.08

### Phusion PCR & Gel extraction

C, G

### Digestion

C, G

### Ligation

C, G

✖

## 2022.08.09

### Transformation

C, G (pSB1A3 in DH5α)

pSB3K3 in BL21

✖

## 2022.08.10

### Digestion & Gel extraction

pSB1A3

### Ligation

C, G (pSB1A3)

✖

## 2022.08.11

### Transformation

C, G in DH5α

### Plasmid purification

C-1, C-2, C-3, C-4

### Digest check

C-1, C-2, C-3, C-4

### SDS-PAGE (Protein volume)

The 2, 4, 6, 8, 10, 12, 14, 16 hr from each: E2-D-1, E2-D-2, E2-F-1, E2-F-2

✖

## 2022.08.12

### Colony PCR

Eight plants from C (8/9 product)
Four plants from each C, G (8/11 product)

### Colony PCR [Bioart]

BFP, YFP, GFP, RFP (pSB1C3 in DH5α)

✖

## 2022.08.13

### Plasmid purification

C-4, C-7, G-1, G-2, G-3

### Digesst check

C-4, C-7, G-1, G-2, G-3

### Colony PCR [Bioart]

Two from GFP: G-5, G-6
Six from BFP, YFP, RFP: B-5~10, Y-5~10, R-5~10

✖

## 2022.08.14

### Digest check

G-1, G-2, G-3

✖

## 2022.08.15

### Colony PCR

8/9 C: 15, 16, 17, 18, 19, 20
8/11 C: 5, 6, 7, 8, 9, 10
8/11 G: 5, 6, 7, 8, 9, 10, 11, 12

✖

## 2022.08.16

### Transformation

A, B in BL21

✖

## 2022.08.17

### SDS-PAGE (Protein concentration)

Expression 2

### Colony PCR

A, B

✖

## 2022.08.18

### Expression

E3-C, E3-A-3, E3-A-4, E3-B-3, E3-B-4

### Plasmid purification

E3-A-3, E3-A-4, E3-B-3, E3-B-4

### Digest check

E3-A-3, E3-A-4, E3-B-3, E3-B-4

✖

## 2022.08.19

### Ligation

Backbone: pSB1A3
Insert: G

### Digest check & Gel extraction

C-6, C-16

✖

## 2022.08.24

### Phusion PCR & Gel extraction

G

### Colony PCR

pSB1A3 (Control)

For start unit: pSB1A3, A, B

### Transformation

A, B in BL21

### Digestion

G

### Ligation

G

✖

## 2022.08.25

### Expression

E4-C, E4-A-3, E4-A-4, E4-B-3, E4-B-4

### Digest check

E4-A-3, E4-A-4, E4-B-3, E4-B-4

### Colony PCR

G

✖

## 2022.08.26

### Plasmid purification

G

### Wash bacteria cells

E4

### Sonicate

2, 4, 6, 8, 10, 12, 14, 16 hr of E4

✖

## 2022.08.27

### Digest check

G

✖

## 2022.08.28

### Sonicate

1, 3, 5, 7, 9, 11, 13, 15 hr of E4

### SDS-PAGE

Expression 4

✖

## 2022.08.31

### Transformation

DH5α: pSB3K3, A, B

BL21: pSB3K3, A, B

✖

## 2022.09.01

### Colony PCR

pSB3K3, A, B (BL21)

pSB3K3, A, B (BL21)

pSB3K3, A, B (DH5α)

### Transformation

pET-30a in DH5α

### Expression

E5-Control, E5-A (pSB3K3)

✖

## 2022.09.02

### Colony PCR

pET-30a (DH5α)

### Plasmid purification

E5-Control-2, E5-Control-3, E5-A-4

A-4, A-8, B-4, B-5

pET-30a-1, pET-30a-2

✖

## 2022.09.05

### Digest check

E5-A-4, A-4, A-8, B-4, B-5

### Digestion

pET-30a-1, pET-30a-2

✖

## 2022.09.06

### Gel extraction

pET-30a-1, pET-30a-2

### Ligation

C, D, E, F (pSB3K3)

### Taq PCR

A, B, C, D, E, F, G

✖

## 2022.09.07

### Transformation

C, D, E, F (pSB3K3, DH5α)

D, F (pSB3K3, BL21)

### Phusion PCR & Gel extraction

A, B, C, D, E, F, G

### Digestion

A, B, C, D, E, F, G (pET-30a)

### Colony PCR

Control, D, F (pSB3K3, BL21)

✖

## 2022.09.08

### Colony PCR

C, D, E, F (pSB3K3, DH5α)

### Expression 6

E6-Control, E6-F-2, E6-F-3

### Digestion & Gel extraction

pET-30a-3, pET-30a-4

### Plasmid purification

E6-F-2, E6-F-3

### Digest check

E6-F-2, E6-F-3

### Transformation

N, O, P

###

✖

## 2022.09.09

### Ligation

A, F, G (pET-30a)

### Digestion

pET-30abackbone

### Wash bacterial cells & Sonicate

Expression 6

✖

## 2022.09.11

### Transformation

A, F, G (pET-30a, DH5α)

pSB3K3, BL21 plasmid

✖

## 2022.09.12

### Phusion PCR & Gel extraction

pET-30a

pSB3K3

### Colony PCR

O, P

### Transformation

N, P

### Colony PCR electrophoresis check

N, P

✖

## 2022.09.13

### Colony PCR electrophoresis check

P

### Transformation

I, J, Q, K, G

✖

## 2022.09.14

### Digest check

C-2, D-2, E-3, F-2 (pSB3K3)

### Gel extraction

pET-30a

### Colony PCR

D, E (pSB3K3, DH5α)

### Ligation

A, B, C, D, E, F, G (pET-30a)

### Transformation

L, J, I

✖

## 2022.09.15

### Colony PCR

L, J, I

✖

## 2022.09.16

### Plasmid Extraction

O

✖

## 2022.09.17

### Colony PCR

A, C, D, E, F, G (pET-30a), L

### Expression 7

E7-Control, E7-F-3, E7-F-4 (pSB1A3, BL21)

✖

## 2022.09.18

### Expression 8

E8-Control, E8-F-3, E8-F-4 (pSB1A3, BL21)

### Digest check

E8-F-3, E8-F-4

### Transformation

O

### Plasmid Extraction

L

### Colony PCR

O

✖

## 2022.09.19

### Digest check

A-3, C-1, C-3, E-2, E-5, F-4, G-2 (pET-30a, DH5α), L

### Transformation

pSB3K3 in BL21

✖

## 2022.09.20

### Expression 9

E9-Control, E9-F-3, E9-F-4 (pSB1A3)

### Colony PCR

O

✖

## 2022.09.21

### Dialysis

E9-Control, E9-F-3, E9-F-4

### Digest check

A-9, A-16 (pET-30a, DH5α), O

### Plasmid Extraction

O, R

✖

## 2022.09.22

### Digest check

A-9, A-16 (pET-30a, DH5α), O, R

✖

## 2022.09.23

### Colony PCR

pSB3K3

### Expression 10

E10-Control-1a3, E10-Control-3k3, E10-F

### Digest check

O

✖

## 2022.09.24

### Transformation

A pSB1A3 to BL21

A pSB3K3 to BL21

### Functional test 1&2

F

### Colony PCR

pSB1A3 BL21

A

### Transformation

R, L

✖

## 2022.09.25

### Expression 11

E11-Control, E11-A

### Digestion

pSB3K3

### Phusion PCR

G

✖

## 2022.09.26

### Plasmid purification

A

### Digest check

A

### Transformation

A, G

✖

## 2022.09.27

### Digest check

A

### Functional test 3

Control, F

### Colony PCR

Control-pSB1A3, Control-pSB3K3, F, G

✖

## 2022.09.28

### Expression 12

E12-Control-1a3, E12-Control-3k3, E12-F

### Functional test 4

F

### Colony PCR

R, L

✖

## 2022.09.29

### Bacteria Culturing

R, L

### Plasmid Extraction

R, L

### Digesst Check

R, L

### Expression

R, L

✖

## 2022.09.30

### Colony PCR

E, G

### Expression 13

Control-3k3, F

### Expression 14

Control-1a3, Control-3k3, E

### BioArt Protein Mixing

✖

## 2022.10.01

### Digest check

E

✖

## 2022.10.02

### Colony PCR

pSB3K3, F

✖

## 2022.10.03

### Expression 15

Control-3k3, F

✖

## 2022.10.04

### Expression 16

Control-3k3, F

✖

## 2022.10.08

### Expression 17

Control-3k3, F

A: His Ubx

B: His EGFP Ubx

C: PaPilA Ubx

D: PaPilA 1-61 Ubx

E: His Y4 mRFP Y293Y296

F: His Y167 mRFP Y240

G: K592100 (2022 kit, plate 1, well 17O)

H: K592024 (2021 kit, plate 1, well 17C)

I: E1010 (2021 kit, plate 3, well 12M)

J: I13500 (2021 kit, plate 6, well 21E)

K: I13500 (2021 kit, plate 2, well 16P)

L: J04450 (2021 kit, plate 6, well 4C)

M: K1033900 (2021 kit, plate 6, well 5N)

N: E1010 (2019 kit, plate 6, well 12M)

O: B0034 (2019 kit, plate 6, well 1M)

P: I13500 (2019 kit, plate 2, well 16P)

Q: I13500 (2019 kit, plate 6, well 21E)

R: K741002 (2019 kit, plate 2, well 17M)

S: K1033900 (2019 kit, plate 6, well 5N)

## 05.09 - 05.15

**Learn more about Ubx:** We want to make elastic fibers that can conduct electricity, finding that the protein Ubx could meet our needs. So we have an in-depth understanding of this protein, including its own characteristics and what kind of protein it can fuse with.

**Search for conductive proteins:** We searched for many kinds of conductive fibers, and decided to use pilA with better conductivity to fuse with Ubx to meet our thought, which we confirmed the production conditions of pilA and optimized its sequence.

**Find other applications:** We want to expand the application in other directions and try to make our fibers have various functions, so our goal is to find more usable proteins.

## 05.16 - 05.22

**Learn Ubx spinning method:** We are looking for the spinning method of Ubx, and the most suitable temperature and conditions for its spinning.

**A new application—antimicrobial peptides(AMPs):** We want to connect AMPs(antimicrobial peptides) to aptamer and then combine them with Ubx to make fibers with the ability of killing specific bacteria. However, the necessity of using aptamers is questionable, coupled with its difficult design. Therefore ,we decided to use only antimicrobial peptides for binding to Ubx.

## 05.23 - 05.29

**Screening suitable AMP candidates:** We searched for various AMPs and compared their differences to find out candidates fitting our needs.

**Find the proteins and conditions that help wound healing:** We want to start with wound dressings ,checking the ingredients of wound dressings currently on the market to know the conditions that aid wound healing.Beside,we attempt to improve the disadvantages of currently marketed wound dressings by adding these proteins to our fibers.

## 05.30 - 06.05

**Find other applications:** Moving on to other applications, we not only checked the scaffolding conditions for cell growth during tissue engineering, but found chelates that can bind to heavy metals, hoping to be used for water filtration and other purposes.

**Meeting with Professor Ming-Chia, Li:** We have had a discussion with Professor Ming Chia Li, who specializes in biomaterials. The professor gave us many suggestions and new ideas. Furthermore, We also exchanged knowledge about materials, and the professor allowed us to go into his lab for tensile testing of materials later.

## 06.06 - 06.12

Optimize the sequence of pilA and AMP

Plan the experimental process

Find other applications

## 06.13 - 06.19

Confirm appropriate conditions of candidate proteins in human body

Design the functional test of each protein and optimize the sequence

## 06.20 - 06.26

To know what affects protein conductivity

Design detailed experiment process and determine plasmid species

## 06.27 - 07.03

Follow-up experiment process design

## 07.04 - 07.10

Previous protein had problems with functional testing

Find another proteins that are easy to conduct functional test

## 07.11 - 07.17

New functional protein—lipase

## 07.18 - 07.24

Follow-up experiment process design

## 05.09 - 05.14

Learn MATLAB

## 05.16 - 05.22

Searching for softwares for protein structure prediction

## 05.23 - 05.29

Searching parameters for RFP expression

## 05.30 - 06.05

Simulation of how the Ubx protein and E.pili protein connect together through SWISS-MODEL

## 06.06 - 06.12

Learn MATLAB SimBiology

Expression Model I

* RFP biobrick
* derive formula
* find parameters

## 06.13 - 06.19

Expression Model II

* GFP biobrick
* derive formula
* find parameters

Expression Model III

* RFP + GFP biobrick
* derive formula

## 06.20 - 06.26

Sorting all data (parameters)

## 06.27 - 07.03

Learn Autodock, Materials Studio

## 07.04 - 07.10

Design verification experiment for expression models

Searching 3D printing models from previous teams

Meeting with professor Ming-Chia, Li

## 07.11 - 07.17

Protein structure prediction

* predict the structure of Ubx with AlphaFold
* label binding sites in fusion proteins with Pymol

## 07.18 - 07.24

Predict the structure of Ubx with I-TASSER

Derive formulas of hydrogel elasticity

Stress analysis with Autodesk Inventor

## 07.25 - 06.31

Meeting with professor Jhih-Wei, Chu

## 08.01 - 08.07

Model plan adjustments

* delete elasticity model
* maintain expression and protein structure model

Protein mixing model

* predict the fluorescence brightness over time

## 08.08 - 08.14

Expression model

* expression induced with different concentrations of IPTG vs the number of proteins

Protein mixing model

* wavelength turn to RGB formula

## 08.15 - 08.21

Protein structure prediction

* I-TASSER: confidence score checking

Binding region prediction (ANCHOR algorithm)

Protein mixing model

* design a Scaffold simulating model to change normal picture into fluorescence

## 08.22 - 08.28

Expression model: experimental verification design

* design biobrick
* design experiment steps
* checking protocol

## 02.29 - 09.04

Expression model: experimental verification design

Molecular dynamics simulation

## 09.05 - 09.11

Expression model: experimental verification

Protein structure prediction: AlphaFold2

## 09.12 - 09.18

Expression model: experimental verification

Protein mixing model

* convert wavelength to RGB

## 0919 - 09.25

Expression model: experimental verification

Binding region prediction: ANCHOR

* analyzing tyrosine binding ability

Protein mixing model

* scaffold model

## 09.26 - 10.02

Expression model: experimental verification

Binding region prediction: ANCHOR

Protein mixing model

* dealing with experimental data
* run the result of protein mixing model

## 10.03 - 10.09

Protein mixing model

* put the interactive result through time on WiKi
* design interactive result presentation

## 10.10 - 10.12

Tyrosine binding model

## 05.09 - 05.15

Establish future plans for HP events.

## 05.16 - 05.22

Write supporting document in Education Package

## 05.23 - 05.29

Make lecture slides in Education Package

## 05.30 - 06.05

6/02 Online meet-up with Professor Ming-Chia, Li

6/05 Online meet-up with team Jiangnan\_China

Make board game in Education package (Script)

## 06.06 - 06.12

Make board game in Education package

## 06.13 - 06.19

Preparation for The 13th AFOB Regional Symposium (2022 ARS event)

## 06.20 - 06.26

6/26 Attend The 13th AFOB Regional Symposium (2022 ARS event)

## 06.27 - 07.03

6/29 Online meet-up with Professor Yu Yuan, Hsiao

7/03 Online meet-up with team Munich

## 07.04 - 07.10

7/04 Online meet-up with team CUG

7/05 Online meet-up with Huai En, Lu

7/09 Release “Synthetic Biology Classroom” video episode 1

## 07.11 - 07.17

7/12 Online meet-up with team HKUST

7/16 Release “Synthetic Biology Classroom” video episode 2

## 07.18 - 07.24

7/18 Online meet-up with team NCHU

7/23 Release “Synthetic Biology Classroom” video episode 3

## 07.25 - 07.31

7/26 Online meet-up with team KCL

7/30 Release “Synthetic Biology Classroom” video episode 4

## 08.01 - 08.07

8/02 Meet-up with GEMS\_Taiwan

8/04 Second online meet-up with team KCL

8/06 7/12 Release “Synthetic Biology Classroom” video episode 5

8/05 Release workshop poster

Make RPG game in Education Package

## 08.08 - 08.14

8/13 Release “Synthetic Biology Classroom” video episode 6

8/14 Launch BioArt workshop #1

## 08.15 - 08.21

8/20 Release “Synthetic Biology Classroom” video episode 7

Make poster of Taiwan SynBio Conference

## 08.22 - 08.28

8/22,23 Attend Taiwan SynBio Conference

8/25 Launch BioArt speech

8/27 Release “Synthetic Biology Classroom” video episode 8

Make promotion video

## 08.29 - 09.04

8/31 Education event with Taipei First Girls High School

9/1 BioArt workshop with KCIS\_Xiugang

9/3 Release “Synthetic Biology Classroom” video episode 9

Make promotion video

## 09.05 - 09.11

9/8 Education event with KCIS\_Xiugang

## 09.12 - 09.18

9/15 NYCU\_Formosa presentation

Workshop video editing

## 09.19 - 09.25

9/23 Entrepreneur visit in Hung Chou Fiber Industry

Workshop video editing

## 09.26 - 10.02

9/28 Bioart phase #3: artwork sharing

Design merchandise

## 10.03 - 10.09

10/9 Publish BioArt online exhibition

Design merchandise

## 10.10 - 10.12

BioArt online exhibition

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
{% block page\_content %}

## Research

It is shown in the research that Ultrabithorax (Ubx) possessed the unique *in vitro* features, which have given it the advantages of developing versatile protein-based biomaterials. Ubx is biocompatible, biodegradable and non-immunogenetic."[1](#citation1)" Those protein properties have highlighted their possible use in biomedicine. Furthermore, Ubx possesses the ability to self-assemble into nanoscale fiber by forming crosslinking dityrosine bonds. It is suggested that dityrosine-containing motifs are the domains contributing to most of its self-assembly."[2](#citation2)" Lastly, a study shows that Ubx can be readily functionalized with other proteins and retain its structure stability and mechanical properties afterwards."[3](#citation3)"

## Image

In our lab work, our aim is to develop a novel biomaterial with dityrosine containing motifs of protein Ultrabithorax (Ubx). We expected the product to retain the self-assembly and mechanical properties when engineered with functional protein mRFP, which further demonstrated its potential to incorporate diverse functional proteins for wide applications in various fields in future. Therefore, we conducted 3 engineering cycles.

![](https://static.igem.wiki/teams/4377/wiki/cycle/cycle1-active.svg)
![](https://static.igem.wiki/teams/4377/wiki/cycle/cycle2.svg)
![](https://static.igem.wiki/teams/4377/wiki/cycle/cycle3.svg)

## Cycle 1: Ubx

### Design

To obtain insights associated with Ubx protein properties, we created a composite part K4377006. (Figure 1). First, we attached 10x His tag at the N terminal of Ubx in order to purify the protein after expression. The attachment of 10x His tag on N terminal instead of C terminal is for the purpose to further amplify out production of Ubx. Second, we performed codon harmonization to reduce protein incorporation in expressed bacteria. The optimized sequence of codons is expected to demonstrate better expression by our engineered *E. coli*.

![](https://static.igem.wiki/teams/4377/wiki/engineering/fig01.png)

**Figure 1.**Composite part K4377006. This composite part contains constitutive promoter, RBS, and Ubx protein.

### Build

#### Cloning result

We successfully amplified Ubx plasmid in *E. coli* DH5α and transformed into *E. coli* BL21 for expression.

![](https://static.igem.wiki/teams/4377/wiki/engineering/fig02.jpg)

**Figure 2.**Ubx plasmid extracted from *E. coli* BL21 digest check result. M - Marker. 1 - Ubx plasmid extracted from *E. coli* BL21 digest check result, backbone (pSB1A3, 2114bp) / insert (composite part K4377006, 1475bp).

#### SDS-PAGE result

After expression, we conducted SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to verify whether the protein was produced (result in Figure 3).

![](https://static.igem.wiki/teams/4377/wiki/engineering/fig03.jpg	)

**Figure 3.**Composite part K4377006 SDS-PAGE result. M - Marker. 1 - Control, pSB1A3 plasmid. 2 - Ubx plasmid, target band Ubx 42.56 kDa.

### Test

As shown in the SDS-PAGE result, the production was below expectation. We further conducted functional tests, but there was no significant evidence that the protein is able to form dityrosine bonds.

### Learn

After discussion, we decided to focus on finding approaches to achieve better production. Thus, we designed engineering Cycle 2.

Next Cycle

## Cycle 2: Binding Region Prediction

### Design

In cycle 2, we developed approaches to narrow down to dityrosine containing domains among Ubx sequence, as dityrosine cross-linkings are evidenced to drive the assembly of proteins and peptides."[4](#citation4)" In step 1, we used the ANCHOR algorithm to predict the region among Ubx sequences that demonstrate better binding. On the other hand, a paper suggested that the position of lysine will affect the rate of formation of dityrosine bonds."[5](#citation5)" This has further led us to our step 2 - building a model to precisely predict exact formation of dityrosine cross linking among the possible binding sites.

### Build

After the analytics of the ANCHOR algorithm in step 1, we further utilize the results and build a selection model to analyze the distance of lysine and tyrosine. The selection model has further specified the binding probability of tyrosine. Based on the step-wise manner of our design, by inputting a baseline value of binding possibility and the sequence to be analyzed, the model will output a line chart based on the list below.

![](https://static.igem.wiki/teams/4377/wiki/engineering/fig04.png)

**Figure 4.**This is the initial output from ANCHOR algorithm, the first row represents the number and the kind of amino acids in the whole sequence. The second row represents the binding probability, the third row represents whether the amino acid has been chosen as the possible binding region, inside the yellow column, is the sequence of Y167 short peptides, which are defined by Figure 5, and the red column shows the peptide Y167 and its probability.

### Test

![](https://static.igem.wiki/teams/4377/wiki/engineering/fig05.png)

**Figure 5.**According to Figure 4, our script will help you generate and check the value then form a line graph.Upon the baseline, we can see two regions with tyrosine at the peak, which represent Y167 and Y240.

### Learn

According to the result of the ANCHOR algorithm and the model we built, Y167 region and Y240 of the Ubx showed the best probability of forming dityrosine cross linking."[6](#citation6)" Thus, we further engineer Y167 and Y240 in our cycle 3.

Next Cycle

## Cycle 3: Y167 mRFP Y240

### Design

With the result of cycle 2, we chose to further design Y167 and Y240 with mRFP in our cycle 3. We first engineered functional protein mRFP (Part:E1010) with Y167 Y240 short peptides in the attempt to verify the self-assembly function. We chose mRFP because it is easy to observe. Second, we attached 10x His tag at the N terminal of Ubx protein in order to purify our protein after expression. The reason we add 10x His tag at N terminal but not C terminal is to amplify the production of Ubx protein. Lastly, we performed codon harmonization to reduce protein incorporation in expressed bacteria. The optimized sequence of codons is expected to demonstrate better expression by our engineered *E. coli*.

![](https://static.igem.wiki/teams/4377/wiki/engineering/fig06.png)

**Figure 6.**Composite part K4377009. This composite part contains a constitutive promoter, RBS, 10x His tag, mRFP, and two functional domains of Ubx protein.

### Build

#### Cloning result

With the same process of building Composite part K4377006, we successfully built Composite part K4377009.

![](https://static.igem.wiki/teams/4377/wiki/engineering/fig07.jpg)

**Figure 7.**Y167 mRFP Y240 plasmid extracted from *E. coli* BL21 digest check result. M - Marker. 1 - Y167 mRFP Y240 plasmid extracted from *E. coli* BL21 digest check result, backbone (pSB1A3, 2114bp) / insert(composite part K4377009, 1066bp).

#### SDS-PAGE result

After expression, we conducted SDS-PAGE to verify the protein was produced (result in Figure 8).

![](https://static.igem.wiki/teams/4377/wiki/engineering/fig08.jpg)

**Figure 8.**Composite part K4377009 SDS-PAGE result. M - Marker. 1 - Control, pSB1A3 plasmid. 2 - mRFP control, E1010 on pSB3K3, target band mRFP 25.4 kDa. 3 - Y167 mRFP Y240 plasmid, target band Y167 mRFP Y240 30.89 kDa.

### Test

#### Solubility of Y167 mRFP Y240

The pellet of Y167 mRFP Y240 plasmid remained red after ultrasonic disruption and centrifuged in PBS buffer. It is assumed that Y167 mRFP Y240 might be insoluble. Therefore, we conducted SDS-PAGE to the amount of Y167 mRFP Y240 in the pellet and supernatant respectively.

![](https://static.igem.wiki/teams/4377/wiki/engineering/fig09.jpg)

**Figure 9.**Solubility of Y167 mRFP Y240 test. M - Marker. 1 - Control, pSB1A3 plasmid in PBS buffer. 2, 3 - Y167 mRFP Y240 plasmid pellet in PBS buffer, target band Y167 mRFP Y240 30.89 kDa. 4 - Control, pSB1A3 plasmid supernatant in PBS buffer. 5, 6 - Y167 mRFP Y240 plasmid supernatant in PBS buffer, target band Y167 mRFP Y240 30.89 kDa.

The result showed the pellet contains more amount of Y167 mRFP Y240 than supernatant in the PBS buffer. In order to obtain more amount of Y167 mRFP Y240, we ultrasonic disrupted Y167 mRFP Y240 pellet and control (pSB1A3 plasmid pellet) in 8M Urea (high concentration chaotropic agent). After disruption, we centrifuged the sample and separated the supernatant to dialyze into Tris Base buffer and ran SDS-PAGE (result in Figure 10).

![](https://static.igem.wiki/teams/4377/wiki/engineering/fig10.jpg)

**Figure 10.**Y167 mRFP Y240 in Tris Base buffer. M - Marker; 1 - Control, pSB1A3 plasmid supernatant in Tris Base buffer. 2, 3 - Y167 mRFP Y240 plasmid supernatant in Tris Base bufferTrisbase buffer, target band Y167 mRFP Y240 30.89 kDa.

Also, we measured the mRFP intensity of the supernatant in both PBS and Tris Base buffer (result in Figure 11).

![](https://static.igem.wiki/teams/4377/wiki/engineering/fig11.jpg)

**Figure 11.**mRFP intensity of the supernatant in both PBS and Tris Base buffer.
As Figure 11 shows, mRFP intensity of the supernatant in Tris Base buffer is way higher than the one in PBS buffer, as a result, we conduct the same process for further experiments.

As Figure 11 shows, mRFP intensity of the supernatant in Tris Base buffer is way higher than the one in PBS buffer. The result shows that mRFP (as functional protein) remains its function after it is fused with two short peptide areas. Then, we conducted the same process for further experiments.

#### Functional Test

To prove that our protein crosslinks with dityrosine bonds. We confirmed the formation of dityrosine bonds from FT/IR and CD measurements of our sample. Later, we explored the application of our biomaterial. Viscosity test results showed that our sample has potential to be bioink. We later 3D printed our sample and acquired the ideal outcome.

![](https://static.igem.wiki/teams/4377/wiki/engineering/fig12.jpg)

**Figure 12.**Macroscopic testing with different concentration of H2O2 and EDTA-Fe

![](https://static.igem.wiki/teams/4377/wiki/engineering/fig15.png)

**Figure 13.**Dityrosine forming process

We used FT/IR-4700 to analyze additional and reduced bondings before and after processing with EDTA-Fe and H2O2. Data proves crosslinking reaction came from dityrosine bond formation with the obvious decrease in C-H bonds. (result in Figure13.)

![](https://static.igem.wiki/teams/4377/wiki/engineering/fig13.png)

**Figure 14.**FT/IR graph of our protein

We did measurement of viscosity for our protein sample and confirmed its shear thinning behavior, which shows high potential for the future development of bioinks for 3D printing.

![](https://static.igem.wiki/teams/4377/wiki/engineering/fig14.png)

**Figure 15.**FT/IR graph of our protein. (a) Viscosity vs. shear rate. (b)The result of 3D printing.

### Learn

Through functional tests, we had successfully achieved our goal of creating the biomaterial that can crosslink and preserve the function of functional protein fusion with them. It is concluded that our biomaterial has the potential to successfully incorporate diverse functional proteins for wide applications in various fields.

## Reference

1. Pavlopoulos, A., & Akam, M. (2011, February 15). Hox gene ultrabithorax regulates distinct sets of target genes at successive stages of drosophila haltere morphogenesis. Proceedings of the National Academy of Sciences of the United States of America.
2. Alexandra M. Greer, Zhao Huang, Ashley Oriakhi(2009, March 18). The Drosophila Transcription Factor Ultrabithorax Self-Assembles into Protein-Based Biomaterials with Multiple Morphologies Biomacromolecules 2009 10 (4),829-837
3. Tsai, S.-P., Howell, D.W., Huang, Z., Hsiao, H.-C., Lu, Y., Matthews, K.S., Lou, J. and Bondos, S.E. (2015), The Effect of Protein Fusions on the Production and Mechanical Properties of Protein-Based Materials. Adv. Funct. Mater., 25: 1442-1450.
4. Fang J, Mehlich A, Koga N, Huang J, Koga R, Gao X, Hu C, Jin C, Rief M, Kast J, Baker D, Li H. Forced protein unfolding leads to highly elastic and tough protein hydrogels. Nat Commun. 2013;4:2974. doi: 10.1038/ncomms3974. PMID: 24352111; PMCID: PMC3983047.
5. Constancio Gonzalez-Obeso, Fredrik G. Backlund, and David L. Kaplan Biomacromolecules 2022 23 (3), 760-765DOI: 10.1021/acs.biomac.1c01192
6. Howell DW, Tsai SP, Churion K, Patterson J, Abbey C, Atkinson JT, Porterpan D, You YH, Meissner KE, Bayless KJ, Bondos SE. Identification of multiple dityrosine bonds in materials composed of the Drosophila protein Ultrabithorax. Adv Funct Mater. 2015 Oct 7;25(37):5988-5998. doi: 10.1002/adfm.201502852. Epub 2015 Aug 31. PMID: 28725173; PMCID: PMC5513195.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block page\_content %}

## Protocols

General Protocols

Bacteria Growth and Processing Protocols

Protein Processing Protocols

Cross-Linking Reaction Protocols

Material Testing Instrument

✖

### General Protocols

[Download](https://static.igem.wiki/teams/4377/wiki/protocols/general-protocols.pdf)

✖

### Bacteria Growth and Processing Protocols

[Download](https://static.igem.wiki/teams/4377/wiki/protocols/bacterial-growth-and-processing-protocols.pdf)

✖

### Protein Processing Protocols

[Download](https://static.igem.wiki/teams/4377/wiki/protocols/protein-processing-protocols.pdf)

✖

### Cross-Linking Reaction Protocols

[Download](https://static.igem.wiki/teams/4377/wiki/cross-linking-reaction-protocols.pdf)

✖

### Material Testing Instrument

[Download](https://static.igem.wiki/teams/4377/wiki/protocols/material-testing-instrument.pdf)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block page\_content %}

## Overview

To produce and verify the functionality of our protein, we first built up a gene construct and then conducted SDS-PAGE to confirm that our protein was correctly produced. To prove that our protein crosslinks with dityrosine bonds. We confirmed the formation of dityrosine bonds from FT/IR and CD measurements of our sample. Later, we explored the application of our Ultrabithorax (Ubx). Viscosity test results showed that our sample is a potential bioink. We later 3D printed our sample and acquired the ideal outcome.

## Mechanism Introduction

Our sequence consists of Y167 Y240 region and mRFP. mRFP was aimed to prove protein keeps its functionality even after crosslinking.

![](https://static.igem.wiki/teams/4377/wiki/results/fig01.png)

**Figure 1.**Tyrosine locations in composite part K4377009 amino acid sequence. (Sequences marked in blue are Y167 Y240 region.)

Crosslinking can be induced by hydroxyl radicals, forming dityrosine covalent bonds with two tyrosines (Figure 2 ).

![](https://static.igem.wiki/teams/4377/wiki/results/fig02.png)

**Figure 2.**Dityrosine forming process

![](https://static.igem.wiki/teams/4377/wiki/results/fig03.png)

**Figure 3.**Artistic representation of cross-linking

## Gene Construct

### Cloning Result

We successfully amplified Ubx plasmid in *E. coli* DH5α and transformed into *E. coli* BL21 for expression.

![](https://static.igem.wiki/teams/4377/wiki/results/fig04.png)

**Figure 4.**The digest check result of Ubx plasmid extracted from *E. coli* BL21. M - Marker. 1 - Ubx plasmid extracted from *E. coli* BL21 digest check result, backbone (pSB1A3, 2114bp) / insert (composite part K4377006, 1475bp)

With the same process of building composite part K4377006, we successfully built composite part K4377009.

![](https://static.igem.wiki/teams/4377/wiki/results/fig05.png)

**Figure 5.**Y167 mRFP Y240 plasmid extracted from *E. coli* BL21 digest check result. M - Marker. 1 - Y167 mRFP Y240 plasmid extracted from *E. coli* BL21 digest check result, backbone (pSB1A3, 2114bp) / insert(composite part K4377003, 1066 bp)

## Protein Expression

### SDS-PAGE Result

After we expressed Ubx plasmid, we conducted SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to verify whether the protein was produced (result in Figure 6).

![](https://static.igem.wiki/teams/4377/wiki/results/fig06.png)

**Figure 6.**Composite part K4377006 SDS-PAGE result. M - Marker. 1 - Control, pSB1A3 pure plasmid. 2 - Ubx plasmid, target band Ubx 42.56 kDa

After the expression of composite part K4377009, we conducted SDS-PAGE to verify the protein was produced (result in Figure 7).

![](https://static.igem.wiki/teams/4377/wiki/results/fig07.png)

**Figure 7.**Composite part K4377009 SDS-PAGE result. M - Marker. 1 - Control, pSB1A3 plasmid. 2 - mRFP control, E1010 on pSB3K3, target band mRFP 25.4 kDa. 3 - Y167 mRFP Y240 plasmid, target band Y167 mRFP Y240 30.89 kDa

## Functional Test

In order to prove that our peptide monomers form feasible cross linkages, we used FT/IR-4700, J-1700 Circular Dichroism Spectrophotometer to examine whether our material forms dityrosine bonds. Finally, to demonstrate our product as a potential 3D printing material, we measured viscosity of our product with Viscometer and further confirmed shear thinning behavior of our material.

### Crosslinking Reaction

According to the paper, Fenton reaction increases the dityrosine bonds in the hydrogels but has little effect on the rheological or mechanical properties.
Therefore, we used H2O2 and EDTA-Fe to prepare our materials through oxidation of tyrosine residues in our protein-based material, leading to dityrosine crosslinking.

![](https://static.igem.wiki/teams/4377/wiki/results/fig08.png)

**Figure 8.**Fenton reaction for dityrosine formation.

We mix H2O2 and EDTA-Fe in eppendorfs of each concentration for them to react before we add 25 μL of our protein sample. H2O2 were prepared in the following concentrations of 0%, 0.1%, 0.15%, 0.2%. While EDTA-Fe were added by 0μg/μL, 0.5μg/μL, 1μg/μL.

![](https://static.igem.wiki/teams/4377/wiki/results/fig09.png)

**Figure 9.**Macroscopic testing with H2O2 and EDTA-Fe. mRFP on the left and Y167 mRFP Y240 on the right.

### FT/IR

To further confirm the existence of dityrosine bonds in our product, we used FT/IR-4700 to analyze additional and reduced bondings before and after crosslinking. As shown in the reaction, C-H bond is reduced and C=C bonds could be observed when dityrosine bonds form.

![](https://static.igem.wiki/teams/4377/wiki/results/fig10.png)

**Figure 10.**Dityrosine forming process

**Table 1.**Frequency range of C-H and C=C in aromatic rings

| Bond | Type of Compound | Frequency Range, cm-1 | Intensity |
| --- | --- | --- | --- |
| C-H | Aromatic rings | 690-900 | Strong |
| C=C | Aromatic rings | 1500-1600 | Variable |

![](https://static.igem.wiki/teams/4377/wiki/results/fig11.png)

**Figure 11.**(a) Transmittance at 875 cm-1 of sample processed with 10μg/μL EDTA-Fe and different concentrations of H2O2 . “Before'' represents samples without crosslinking. (b) Transmittance of sample processed with 20μg/μL EDTA-Fe and different concentrations of H2O2 . “Before'' represents samples without crosslinking.

Figure 11 shows our result with FT/IR, at 875 cm-1,where the frequency range of the C-H bond, the transmittance changed significantly after crosslinking. C-H bond is reduced when dityrosine bonds form. Thus, higher transmittance represents a greater amount of dityrosine bonds.

Based on results from Figure 9 and Figure 11, we took each sample that contains the highest amount of dityrosine bonds from both precipitation and homogeneous groups for future tests. Sample 1 (adding 10μg/μL EDTA-Fe, 0.2% H2O2) from precipitation group and Sample 2 (adding 10μg/μL EDTA-Fe, 0.1% H2O2) from homogeneous group.

![](https://static.igem.wiki/teams/4377/wiki/results/fig12.png)

**Figure 12.**(a) FT/IR graph of sample 1 before and after processing with 10μg/μL EDTA-Fe and 0.2% H2O2 (b) FT/IR graph of sample 2 before and after processing with 10μg/μL EDTA-Fe and 0.1% H2O2

Figure 12 shows the significant influence of EDTA-Fe and H2O2 on 875 nm(C-H bond) transmittance. (a) Sample transmittance increased 12.22% after adding reagent. (b) Sample transmittance increased 6% after adding reagent. Data above proves crosslinking reaction came from dityrosine bond formation with the obvious decrease in C-H bonds.

It's observed from Figure13, 14 that C-H bonds were reduced at 690-900 nm, and that C=C bonds were produced at 1500-1600 nm. Based on data acquired from FT/TR measurements, we have successfully proved the crosslinking reaction came from dityrosine bond formation.

![](https://static.igem.wiki/teams/4377/wiki/results/fig13.png)

**Figure 13.**FT/IR graph of mRFP before and after processing with (a) 10μg/μL EDTA-Fe and 0.2% H2O2 (b) 10μg/μL EDTA-Fe and 0.1% H2O2

To confirm Y167 mRFP Y240 crosslinking surely occurs from dityrosine bonds in Y167Y240 region and not mRFP protein, we did FT/IR measurements of sole mRFP. Observed from acquired data, results barely changed after adding different concentrations of EDTA-Fe and H2O2 , showing the change of C-H bonds in our protein sample did come from tyrosines in protein residues Y167Y240 region and not mRFP.

### Circular Dichroism

In addition to observing changes in bonding, we also measure absorbance of Sample 1 with J-1700 Circular Dichroism before and after crosslinking. After crosslinking, redshift occurs in the band of absorption values. Showing that two benzene ring bonds together after dityrosine bond formation. Electron delocalization of benzene rings leads to the change of wave pattern in graphs.

![](https://static.igem.wiki/teams/4377/wiki/results/fig14.png)

**Figure 14.**Absorbance graph of Y167 mRFP Y240 before and after processing with (a) 10μg/μL EDTA-Fe and 0.2% H2O2 (b) 10μg/μL EDTA-Fe and 0.1% H2O2

Observed from Figure 14, redshift occurs after reagents (10μg/μL EDTA-Fe and 0.2% H2O2) were added to Sample 1 and absorbance was higher than unprocessed samples. This could be the consequence of scattering caused by precipitation. Sample 2 (adding 10μg/μL EDTA-Fe and 0.1% H2O2) decreased in 275 nm absorbance and increased in 310 nm absorbance. Showing that electrons shifting from dityrosine formation changed the wave pattern.

Based on our Circular Dichroism result, we chose Sample 2 (which shows better results from absorption values) to excite our protein sample with 275 nm UV light, observing our sample giving out red and blue fluorescence at the same time. Red fluorescence came from mRFP and blue fluorescence came from dityrosine bond formation.

![](https://static.igem.wiki/teams/4377/wiki/results/fig15.png)

**Figure 15.**Fluorescence of our sample (a) before and (b) after processing with EDTA-Fe and H2O2 at 275 nm excitement.

### Viscometer

We envision our product to be a biomaterial of great use. In professor Ming-Chia, Li's lab, we did measurement of viscosity for our protein sample and confirmed its shear thinning behavior. This proves that our Y167 mRFP Y240 biomaterial has great potential as 3D printing materials.

![](https://static.igem.wiki/teams/4377/wiki/results/fig16.png)

**Figure 16.**Viscosity vs. Shear rate of Y167 mRFP Y240

The result of viscosity vs. shear rate shows that the viscosity decreases when the shear rate increases, illustrating the properties of shear thinning, which shows high potential for the future development of bioinks for 3D printing.

### Operation of 3D printer

To prove that our Ubx biomaterial can serve as a bioink for 3D printing, we learned to operate 3D printers from professor Ming-Chia, Li and printed a graph as we expected. View more details in [Proof of Concept](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27proof-of-concept%27%29%20%7D%7D). Result shows that the Y167 Y240 region separated from Ubx is a successful bioink material. Aside from cross linking monomers, Ubx biomaterial also ensures functional proteins maintain their functionality after crosslinking. Providing a brand new and advanced biomaterial with great potential in medical, industrial, and agricultural fields.

## Reference

1. Choi, J., McGill, M., Raia, N. R., Hasturk, O., & Kaplan, D. L. (2019). Silk hydrogels crosslinked by the fenton reaction. Advanced healthcare materials, 8(17), 1900644.

{% endblock %}