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

Franek

Tasks:

• Transformation of competent cells with BBa_K177024 and BBa_K177025

Methods:

• Chemocompetent E. coli cells from Top10 strain were transformed according to our standard procedure with either BBa_K177024 or BBa_K177025

• Tranformants with BBa_K177024 were plated on 18 plates with LB, agar-agar and IPTG

• Tranformants with BBa_K177025 were plated on 18 plates with LB, agar-agar and 0.2 % arabinose.

Results:

• Positive selection will be made according to fluorescence under UV light

Making of the RBS-cI

Jarek

Tasks:

• Separation of digested DNA in 0,8% agarose gel.

Cloning of p53 coding sequence

Marcin

Task 1:

• Transformation of chemocompetent strain of E. coli by plasmid containing p53 sequence

Methods

• The protocol of transformation has not been changed, except of giving 10 µl of ligated plasmid solution to the bacteria. If you want to see detailed procedure go here
• Bacteria was plated on the medium containing ampicillin, X-Gal and IPTG

Task 2: Restriction digest of pKS vector for future aplications:

Methods:

• Reaction mixture composition:

  2 μl pKS plasmid
  1 μl XbaI (Fermentas)
  1 μl SmaI (Fermentas) 
  2 μl Buffer Tango (Fermentas)
  14 μl MQ water

Program:

1. 37°C - 8 hours
2. 65°C - 15 minutes

Assembly of endosomal detection operon

Marcin

Task 1:

• Breed bacteria to isolate plasmid containing biobricks essential for build the operon:

BBa_B0032

BBa_c0040

BBa_C0051

BBa_E0032

Methods:

1. Prepare LB medium with ampicillin
2. Add 3.5 ml of the medium to the probes
3. Add one bacterial colony to each probe
4. Breed the bacteria about 10 hours

Cloning of the mgtc promoter into the pKSII+ plasmid

Kamil

Tasks:

• Ligation verification
• Bacteria transformation

Methods:

• Correctness of the ligation was verified with gel electrophoresis on 1% agarose gel.

• A 200μl batch of chemocompetent bacteria was transformed with 5μl of ligation mix and incubated overnight on petri dishes containing LB medium supplemented with ampicilin, X-gal and IPTG.

Results:

Electrophoresis results:

From left:

1. GeneRuler DNA Ladder Mix #SM0333 (Fermentas)
2. ligation

NOTES: The roughly 1,5kb fragment (b) represents the digested plasmid, the bearly visible fragment (c) weighing about 0,5kb is the digaested mgtc promoter and the (a) band represents the anticipated closed plasmid (dragged back on the electrophoresis due to it's circular natuire).

Conclusions:

• The new ligation method has proven to be successful and the sample is good to be used for transformation.
  

Construction of K177012 operon1_part2

Ania

Tasks:

• Set up a liquid culture from traqnsformants containing BBa_C0012 - lacI repressor on the pSB1A2 ampicillin resistant plasmid.

• Alkaline lysis of bacterial cultures to obtain plasmids containing following bricks:

BBa_C0012 - lacI repressor on the pSB1A2 ampicillin resistant plasmid.

• Digest of BBa_C0012 - lacI repressor on the pSB1A2 ampicillin resistant plasmid (XbaI/PstI = prospective insert)
• Digest of BBa_B0032 - RBS.3 on the pSB1A2 ampicillin resistant plasmid (SpeI/PstI = prospective vector)

Results:

• BBa_C0012 - lacI repressor and BBa_B0032 - RBS.3 properly digested.

Isolation of BioBricks from 2008 and 2009 Kit Plates

Results from 13 July 2009

• Isolation of GFP coding device switched on by IPTG - BBa_I763004 was unsuccessful

Tasks:

• Alkaline lysis of bacterial cultures to obtain promoter lambda (cI regulated) with RFP reporter BBa_I763007 (procedure described here)
• Control digestion of BBa_I763007, BBa_E0840, BBa_J07037 and BBa_B0024

Methods

• Digestion of plsmids containing BioBricks by following enzymes (Fermentas) in proper buffers for 2h in 37°C