Experiments

Violacein AND gate

The original design for the AND gate was to use an igem part T7 polymerase AND gate (BBa_K568004). However this part along with the Vio operon would result a part that is almost 15kb. Our PI noted us of the difficulties of transforming a part of such size an suggested a different AND gate design. His AND gate design was a split vio operon which had few vio genes under control of one promoter and the rest of the genes under the control of a different promoter. Thus, the full violacein pathway is constituted only when both of the promoters are activated.

As described in the Description page, we decided to use light as one of the inputs for violacein production. For the second input, for simplification and reliability, the IPTG inducible lac promoter was used. Gibson assembly was used to connect two addgene plasmids:

Violacein `addgene' vector

The final construct is as follows.

Though it would be the best to include the full violacein pathway, the gibson process would be a lot more complicated. Thus, our PI suggested a proof of concept plasmid which has vioA under the control of pDawn system and vioB and vioE under the control of the IPTG inducible system.

Due to time constraints and the fact that there were no experienced wet lab member in our team, our wet lab instructors Sera Kang and Xiaoyue conducted the assembly and transformation proccess.

The protocols for the experiments can be seen below.

Protocol

A. Prepare pDawn vector with enzyme cut for Gibson Assembly(GA)

- plasmid enzyme cut → check gel → inactivation → clean up

1. Mix all below chemical substances to make total 50 ㎕ solution.

Total	50 ㎕
pDawn	40 ㎕
NdeI	1 ㎕
NotI-HF	1 ㎕
Cutsmart(10x)	5 ㎕
DW	3 ㎕

2. Let mixture in 37℃ for 50 minutes.

3. Check gel with uncut.

4. Put mixture in 65℃ for 20 minutes.

5. Clean up

- 1-1, 1-3 -> 50 ㎕ elution

- 2-1, 2-2, 2-3 -> 100 ㎕ elution

B. Prepare insert sequence "vio ABE" PCR for GA

1. Mix all below chemical substances

Total	25 ㎕
template(1/10)	2 ㎕
reaction buffer	5 ㎕
dNTP	2 ㎕
Primer -Forward	1 ㎕
Primer -Reverse	1 ㎕
Q5 polymerase	0.2 ㎕
DW	13.8 ㎕

* plasmid: pETM6-G6-vioABE-4A6-vioC-3A2-vioD

* primer

(1) vioA(1.4 kb) : vioA_F(55℃), vioA_R(60℃)

(2) vioAB(4.7 kb) : vioA_F(55℃), vioAB_R(53℃)

(3) vioAB(vioABE)(3.5 kb) : vioA_F(55℃), vioABE_mid_R(56℃)

(4) vioBE(vioABE)(2.1 kb) : vioABE_mid_F(56℃), vioABE_R(54℃)

(5) vioABE(5.6 kb) : vioA_F(55℃), vioABE_R(54℃)

2. Turn PCR.

- (1) 56℃ / 58℃ / 60℃ in 1 minute.

- (2), (5) 53℃ / 55℃ / 57℃ in 3 minutes.

- (3) 54℃ / 56℃ / 58℃ in 2 minutes.

- (4) 53℃ / 55℃ / 57℃ in 1 and a half minute.

- (1), (4) 53℃ / 56℃ / 60℃ in 1 and a half minute.

=> Mix and put DPN 1.5 ㎕ each of them.

- (2), (3), (5) 53℃ / 56℃ / 58℃ in 3 minutes.

=> (5) showed 2 bands, so we redo with a little higher temperature.

- (5) again 58℃ / 60℃ / 62℃ in 3 minutes.

=> It showed 2 bands again, and we figured out that vioA_F has overlapped sequence so that it resulted in 2 bands.

Result band of vioA and vioBE

Result band of vioAB

C. Combine vector and insert in order to do GA

1. vioABE

pDawn(vector)	vioAB(vioABE)	vioBE(vioABE)
7.1 kb	3.5 kb	2.1 kb
21 ng/㎕	47 ng/㎕	65 ng/㎕
0.0045 pmol	0.02 pmol	0.047 pmol

(1) Mix vioAB and vioBE with 2 : 1 ratio.

(2)

	1:2	1:3
vector	1/5	1.5/5

(3-1)

Total	20 ㎕
pDawn	5 ㎕
insert	1 ㎕
HiFi master mix	10 ㎕
DW	4 ㎕

(3-2) If insert is 0.03 to 0.2 pmol, follow below table. (This can make total amount in a half)

Total	20 ㎕
pDawn	5 ㎕
insert	1.5 ㎕
HiFi master mix	10 ㎕
DW	3.5 ㎕

2. vioA

pDawn(vector)	vioA(insert)
7.1 kb	1.4 kb
21 ng/㎕	54 ng/㎕
0.0045 pmol	0.058 pmol

(1)

	1:2	1:3
vector	1.5/6.5	1.5/6.5

(2-1)

Total	20 ㎕
pDawn	6.5 ㎕
vioA	1 ㎕
HiFi master mix	10 ㎕
DW	2.5 ㎕

(2-2)

Total	20 ㎕
pDawn	6.5 ㎕
vioA	1.5 ㎕
HiFi master mix	10 ㎕
DW	2 ㎕

3. vioAB

pDawn(vector)	vioAB(insert)
7.1 kb	4.7 kb
21 ng/㎕	30 ng/㎕
0.0045 pmol	0.097 pmol

(1)

	1:2	1:3
vector	4/4	6/4

(2-1)

Total	20 ㎕
pDawn	4 ㎕
vioAB	4 ㎕
HiFi master mix	10 ㎕
DW	2 ㎕

(2-2)

Total	20 ㎕
pDawn	4 ㎕
vioA	6 ㎕
HiFi master mix	10 ㎕

4. control

Total	10 ㎕
pDawn	2.5 ㎕
DW	7.5 ㎕

5. Put all in 50℃ for 15 minutes.

6. transformation (DH5α)

D. vio ABE chemical transformation

1. Mix competent cell 50 ㎕ and plasmid(pDawn_vio ABE) 2 ㎕.

2. Incubate ice for 30 minutes.

3. Incubate it in 43℃ for 40 seconds.

4. Incubate it in ice for 5 minutes.

5. Mix LB 1ml.

6. Incubate in 37℃ for 1 hour.

7. Centrifuge 13,500 rpm for 1 minute.

8. Discard supernatant 800ul and resuspense leftover.

9. Spread each of 100 ㎕ in LB/KAN plate.

10. Separate each plate with light, and without light. So wrap with tinfoil plate without light.

11. Incubate them in 37℃ through overnight.

Results

- We made vector and insert sequence that include vio genes into a single plasmid through Gibson assembly.

- By inserting the plasmid into E. coli through transformation, it was possible to create a strain having vioABE that we wanted. This strain was observed to form colonies while growing them in medium.

- However, the results were not promising since there were no visible difference among the two plates. It may be that the light of the incubator was not strong enough to induce a response or maybe the product was not enough to be differentiated with the naked eye. This experiment will need further debugging

- We can get some pictures after transformation that we gained the strain we wanted.