Creating devices to test promoters in E. coli strains (devices BBa_K177024 and BBa_K177025)

Franek

Tasks:

• Selecting clones with BBa_K177024 and BBa_K177025

Methods:

• Tranformants with BBa_K177024 and BBa_K177025 clones were recognised due to the fluorescence under UV light.

• 4 positive BBa_K177024, and 16 BBa_K177025 were found.

• Each clone was transferred to new plate with LB, agar-agar and IPTG or 0.2 % arabinose. Liquid cultures were also established.

Results:

• BBa_K177024 and BBa_K177025 were successfully created!

Cloning of the mgtc promoter into the pKSII+ plasmid

Kamil

Tasks:

• Bacteria transformation

Methods:

• A 200μl batch of chemocompetent bacteria was transformed with 15μl of ligation mix and incubated overnight on petri dishes containing LB medium supplemented with ampicilin, X-gal and IPTG.

Results:

The transformation yielded only a couple of blue colonies.

Conclusions:

• The transformation needs to be redone, this time using more of the ligation mix.

Assembly of endosomal detection operon

Marcin

Task 1:

• Isolate plasmids containing the biobricks

Methods:

• Plasmids weren isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described here.
• After the isolation 1 ul of each plasmid solution was diluted to 10 ul and loaded into the 1% agarose gel
• Electrophoresis condition:

voltage - 70V

time - 30 min

• Next the gel was photographed:

Legend:

1-3 - BBa_B0032

4-6 - BBa_c0040

7-9 - BBa_C0051

10-12 - BBa_E0032

Comment:

Isolation of plasmids was successful.

Cloning of p53 coding sequence

Marcin

Task:

• Cloning p53 coding sequence to pKS plasmid

Methods:

• Ligation mixture composition:

  14 μl digested p53 
  1.5 μl digested pKS
  5 μl ligation buffer (Invitrogen)
  1 μl ligase T4

• Duration of ligation was about 20 hours; reaction was conducted in 16 °C (approximately).

Insertion of the pho gene into the pKSII+ plasmid

Kama

• Isolation of plasmid containing an insert was performed with the A&A plasmid mini kit

Notes

• All samples contain empty plasmid

Construction of K177012 operon1_part2

Ania

Tasks:

• Electrophoresis, gel extraction and ligation of:
• BBa_C0012 - lacI repressor on the pSB1A2 ampicillin resistant plasmid (XbaI/PstI = prospective insert)
• BBa_B0032 - RBS.3 on the pSB1A2 ampicillin resistant plasmid (SpeI/PstI = prospective vector)