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From 2014.igem.org

Contents 1 Labwork 1.1 B-Contruction of the fusion protein 1.2 D- Lemon scent 1.2.1 Limonene synthase 1.2.2 pPPS5 1.2.3 pPS3 and pPS4 1.2.4 Limonene synthase 1.2.5 pPS5

Labwork

B-Contruction of the fusion protein

By Hv

We can see that the blue colonies of E.coli are not blue in a simple petri dish with ampicillin. It's a fail. So, to figure our problem we decided to do a sequencing of our plasmids.

We chose 6 colonies from an Xgal-IPTG petri dish:

- 1 completely blue

- 2 blue with a white ring around the blue

- 3 white colony

PS: we found that there is 2 types of blue colonies: completely blue and blue with a white ring around the blue)

We first did a liquid culture in 5 mL of LB.

D- Lemon scent

By Melanie

Limonene synthase

Migration of the PCR done yesterday

photo

well 1= ladder well 2-6 = pPS5 other well = LS PCR As we see, there are nothing on our gel so we conclude that we failed to clone LS in pGMETeasy

pPPS5

the photo in the same as previously: We saw a band but we will check another time by PCR to be sure.

pPS3 and pPS4

Plasmid extraction using the plasmid DNA extraction kit and electrophoresis:

[photo]

We conclude that we have done a good manipulation.

Limonene synthase

As we saw that we don't have anything for LS, We try to do other PCR from the Biobrick (BBA762100). We use two enzyme: Dream taq and Vent taq

component	volume
H2O	42μl
buffer	5μl
dNTPs	1μl
iPS66	1μl
iPS67	1μl
DNA	0.5μl
enzyme	0.5μl

Tubes were placed in PCR machine with the following parameters.

Cycle step	Temperature	Time	Cycle
Initial denaturation	98°C	1 min	1
Denaturation	98°C	15 s	25 - 30
Annealing	52°C	25 s	25 - 30
Extension	72°C	45 s	25-30
Final extension	72°C	10 min	1
Final extension	8°C	hold	1

pPS5

To have more plasmid We do some bacteria culture (pPS5)