Cloning of the mgtc promoter into the pKSII+ plasmid

Kamil

Tasks:

• Transformant selection

Methods:

• White colonies were picked up and transferred to a new petri dish.
• Liquid cultures were established along the way for plasmid purification.
• Both the dish and the liquid cultures were incubated at 37°C overnight.

Cloning of p53 coding sequence

Marcin

Task:

• Transformation of chemocompetent E. coli strain DH5α

Methods:

• Ligation reaction was stopped via thermal inactivation in 80°C for 20 minutes
• Detailed protocol of ligation is described here. The only modification is usage of half volume of ligation mixture prepared 15.07.09

Assembly of endosomal detection operon

Marcin

Task:

• Restriction digest of biobricks

Comment:

Due to obtain set of biobricks which each of them contain RBS and particular coding sequence some of biobricks were digested:

BBa_B0032

BBa_c0040

BBa_C0051

BBa_E0032

Methods:

• Digest of BBa_B0032 using SpeI and PstI
• Reaction mixture composition: 10 μl purified plasmid DNA product, 0.5 μl SpeI (Fermentas),1 μl PstI (Fermentas), 5 μl Buffer Tango (Fermentas), 34 μl MQ water
• Digest of other biobricks using PstI and XbaI
• Reaction mixture composition: 10 μl purified plasmid DNA product, 1 μl XbaI (Fermentas),1 μl PstI (Fermentas), 5 μl Buffer Tango (Fermentas), 34 μl MQ water
• Both reaction were performed overnight (~12 hours) and both of them were subsequently inactivated via heating in 80°C for 20 minutes

Construction of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K177012 K177012] operon1_part2

Ania

Tasks:

• Set up a liquid culture from transformants containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0032 BBa_B0032 - RBS.3] and [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 plasmid.

• Digest of the ligated [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0032 BBa_B0032 - RBS.3] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0012 BBa_C0012 - lacI repressor] on the pSB1A2 ampicillin resistant plasmid with EcoRI and PstI to confirm ligation.

Testing different E. coli strains regarding [http://partsregistry.org/Part:BBa_R0010 lacI] and [http://partsregistry.org/Part:BBa_R0080 AraC] repressors

Franek

Tasks:

• Alkaline lysis of bacterial cultures to obtain [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] and [http://partsregistry.org/Part:BBa_K177025 BBa_K177025] devices

• Transformation of Top10/ BL/ GM/ V1011/ V1012/ TopF'/ DH5a competent cells with [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] and [http://partsregistry.org/Part:BBa_K177025 BBa_K177025]

Methods:

• 2 test tubes with 5ml LB and ampicillin were first inoculated with colonies containing [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] and [http://partsregistry.org/Part:BBa_K177025 BBa_K177025] on [http://partsregistry.org/Part:pSB1A2 pSB1A2] plasmid. The cultures were incubated overnight at 37°C. Alkaline lysis was performed with [http://aabiot.com/products/dna_purification/plasmid_dna/plasmid_mini/protocol_plasmid_mini.pdf PlasmidMini set by A&A Biotechnology]. The pellet from 5 ml of bacteria was used.

• Chemocompetent E. coli cells from Top10/ BL/ GM/ V1011/ V1012/ TopF'/ DH5a strains (prepered earlier by Kuba) 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]

Results:

• Will be determined by cell colonies presence on plates