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Team:Warsaw/Calendar-Main/14 July 2009
From 2009.igem.org
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Creating devices to test promoters in E. coli strains (devices [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] and [http://partsregistry.org/Part:BBa_K177025 BBa_K177025])
Franek
Tasks:
- Transformation of competent cells with [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] and [http://partsregistry.org/Part:BBa_K177025 BBa_K177025]
Methods:
- Chemocompetent E. coli cells from Top10 strain were transformed according to our standard procedure with either [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] or [http://partsregistry.org/Part:BBa_K177025 BBa_K177025]
- Tranformants with [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] were plated on 18 plates with LB, agar-agar and IPTG
- Tranformants with [http://partsregistry.org/Part:BBa_K177025 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
Cloning p53 coding sequence
Marcin
Task 1:
- Transformation of chemocompetent strain of E. coli by plasmid containing p53 sequence
Procedure:
- 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:
digest:
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:
[http://partsregistry.org/Part:BBa_B0032 BBa_B0032]
[http://partsregistry.org/Part:BBa_C0040 BBa_C0040]
[http://partsregistry.org/Part:BBa_C0051 BBa_C0051]
[http://partsregistry.org/Part:BBa_E0032 BBa_E0032]
Methods:
- Prepare LB medium with ampicillin
- Add 3.5 ml of the medium to the probes
- Add one bacterial colony to each probe
- 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:
- GeneRuler DNA Ladder Mix #SM0333 (Fermentas)
- 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 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K177012 K177012] operon1_part2
Ania
Tasks:
- Set up a liquid culture from traqnsformants containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0012 BBa_C0012 - lacI repressor] on the pSB1A2 ampicillin resistant plasmid.
- Alkaline lysis of bacterial cultures to obtain plasmids containing following bricks:
[http://partsregistry.org/wiki/index.php?title=Part:BBa_C0012 BBa_C0012 - lacI repressor] on the pSB1A2 ampicillin resistant plasmid.
- Digest of [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0012 BBa_C0012 - lacI repressor] on the pSB1A2 ampicillin resistant plasmid (XbaI/PstI = prospective insert)
- Digest of [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0032 BBa_B0032 - RBS.3] on the pSB1A2 ampicillin resistant plasmid (SpeI/PstI = prospective vector)
Results:
- [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0012 BBa_C0012 - lacI repressor] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0032 BBa_B0032 - RBS.3] properly digested.
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