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Team:Warsaw/Calendar-Main/15 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:
- Selecting clones with [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] and [http://partsregistry.org/Part:BBa_K177025 BBa_K177025]
Methods:
- Tranformants with [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] and [http://partsregistry.org/Part:BBa_K177025 BBa_K177025] clones were recognised due to the fluorescence under UV light.
- 4 positive [http://partsregistry.org/Part:BBa_K177024 BBa_K177024], and 16 [http://partsregistry.org/Part:BBa_K177025 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:
- [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] and [http://partsregistry.org/Part:BBa_K177025 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:
Samples of plasmids after isolation
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 [http://partsregistry.org/wiki/index.php?title=Part:BBa_K177012 K177012] operon1_part2
Ania
Tasks:
- Electrophoresis, gel extraction and ligation 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)
- [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0032 BBa_B0032 - RBS.3] on the pSB1A2 ampicillin resistant plasmid (SpeI/PstI = prospective vector)
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