Notebook : July 11

Summary:

For régulation system:

• 1.For those transformation products(of yesterday), RBS+LacS+terminator in PSB1C3 plasmid, the liquid culture has been performed for further experiments. The oligopetides for PCR amplification was made bioinformaticly. The transformation for the terminator BBa_B0010 was still fail.
• 2. The plasmid which contains fnr+RBS+LacZ+terminator and fnr+RBS+AmilCP+terminator was extracted. The restiction digest was performeed for them.

For sensor system:

• 3. The selection of bacterian colonies was achieved by analyzing PCR product. The baterian selected are BphR1 c5 and c6; BphR2 c3 and c4; BphA1 c5 and c6,c7,c8.

Lab work

A.aero/anaerobic regulation system:

• BioBrick RBS+LacZ+terminator in plasmid PSB1C3

Transformation for BBa_I732019 terminator

After ong night culture, we observed 2 tiny colonies on the medium. We continued the experiments by performing another liquid culture at 37°C with ampicillin.

Transformation for BBa_B0010 was always no go

Verification the transformation of BBa_I004450 in PSB3K3 by digestion and eletrophoresis

We performed 2 types of digestion:

Simple digestion:

• DNA: 3µl
• Buffe:r 3 µl
• Enzyme: 1µl
• H2O: 23µl
• Total: 30µl

Double digestion:

• DNA: 5µl
• Buffer: 3 µl
• Enzyme: 2µl
• H2O: 20µl
• Total: 30µl

Buffer used:

• Ecor I+ PST I -> orange
• Xho I -> green
• Sac II -> blue
• XhoI+Sac II -> green

After the digestion, we performed a eletrophoresis for verification:

Well 1,7 :XhoI+Sac II Well 2,8 :Sac II Well 3,9 :Ecor I+ PST I Well 4,10 :Xho I Well 5,11 : control no digested gel 0.8%

Estimed size and observed size:

enzyme	estimed size	observed size
Ecor I+PST I	1069bp and 2750 bp	1000bp and 2700bp
Xho I	2976bp+842bp	4000bp
Sac II	3819bp	4000bp
Xho I+Sac II	843bp,616bp,2367bp	1200bp and 2000bp

We confimed the existence of BBa_I04550 in plasmid PSB3K3.

B.PCBs sensor system:

• Construction for BioBrick promoter promoter BphR1, BphR2, BphA1

Colony PCR

From the culture of cloning for promoter BphR1, BphR2, BphA1 which we seeded yesterday, we had chosen 8 colonies in total for further test. And like we did for promoter fnr, we used 8 primers for PCR amplification, they were: BphR1 up/down, BphR2 up/down, BphA1 up/down and Vf/VR.

In order to make clear this large number of PCR tubes, we classified them into 4 lots. they were:
Mix A : promoter BphR1

• buffer go ta:(1X) : 5µl
• MgCL2: 2µl
• dNTP: 1µl
• primers(R1_up/VR or VF/R1_down): 0.125µl
• DNA: 2µl
• Enzyme: 0.25µl
• H2O: about 14.5µl
• total: 25µl

Mix B : promoter BphR2

• buffer go ta:(1X) : 5µl
• MgCL2: 2µl
• dNTP: 1µl
• primers(R2_up/VR or VF/R2_down): 0.125µl
• DNA: 2µl
• Enzyme: 0.25µl
• H2O: about 14.5µl
• total: 25µl

Mix C : promoter BphA1

• buffer go ta:(1X) : 5µl
• MgCL2: 2µl
• dNTP: 1µl
• primers(A1_up/VR or VF/A1_down): 0.125µl
• DNA: 2µl
• Enzyme: 0.25µl
• H2O: about 14.5µl
• total: 25µl

Mix D :

• buffer go ta:(1X) : 5µl
• MgCL2: 2µl
• dNTP: 1µl
• primers(VF/VR): 0.125µl
• DNA: 2µl
• Enzyme: 0.25µl
• H2O: about 14.5µl
• total: 25µl

PCR program:

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