Insertion of the pho gene into the pKSII+ plasmid

Kama

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 in order to amplify the plasmid and extract it from the bacteria.

Comment:

• Accidentally I used the whole DNA samples extracted on Thursday to perform the digestion. I Added the digestion mix to my samples, hence the wrong buffer concentration and improper digestion on Friday. Thinking saves time and money. But there is another reason to justify the repeated plasmid extraction. Now I know that my transformants after all have the correct plasmid so I can do the alkaline lysis on very few samples and use the AA kit to get greater DNA concentration. After all this is my insert for the next ligation, so we need a lot. I know that no one will ever read the notebook up to this point.

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

Franek

Tasks:

• Seting liquid cultures 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:

• 7 test tubes with 5ml LB were inoculated with Top10/ BL/ GM/ V1011/ V1012/ TopF'/ DH5a. The cultures were left for overnight incubation at 37°C.

• 14 test tubes with 5ml LB and ampicillin were inoculated with Top10/ BL/ GM/ V1011/ V1012/ TopF'/ DH5a, containing either [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] or [http://partsregistry.org/Part:BBa_K177025 BBa_K177025] on [http://partsregistry.org/Part:pSB1A2 pSB1A2] plasmid. The cultures were left for overnight incubation at 37°C.

• 7 test tubes with 5ml LB, ampicillin and IPTG were inoculated with Top10/ BL/ GM/ V1011/ V1012/ TopF'/ DH5a, containing [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] on [http://partsregistry.org/Part:pSB1A2 pSB1A2] plasmid. The cultures were left for overnight incubation at 37°C.

• 7 test tubes with 5ml LB, ampicillin and 0,2% arabinose were inoculated with Top10/ BL/ GM/ V1011/ V1012/ TopF'/ DH5a, containing [http://partsregistry.org/Part:BBa_K177025 BBa_K177025] on [http://partsregistry.org/Part:pSB1A2 pSB1A2] plasmid. The cultures were left for overnight incubation at 37°C.

Results:

• Will be determined next day by comparing GFP level in diffrent cultures

Cloning of the mgtc promoter into the pKSII+ plasmid

Kamil

Tasks:

• Plasmid assembly

Methods:

• Plasmid digest mix was prepared as follows: 2μl Tango buffer (Fermentas), 3μl pKSII+ plasmid, 2μl XbaI enzyme, 2μl SmaI enzyme, the solution was topped up with H2O to the final volume of 20 ul.
• mgtc promoter digest mix was prepared as follows: 2μl Tango buffer (Fermentas), 10μl purified gene, 2μl XbaI enzyme, the solution was topped up with H2O to the final volume of 20 ul.
• The digest was kept for 6h (to compensate for the 50% loss in activity of the SmaI enzyme) at 37°C, and then the enzyme was inactivated for 15min. at 80°C.

• The ligation mix was prepared as follows: 10μl of both inactivated digests were mixed together and topped with 2μl of 30% PEG, 2,2μl ligation buffer (containing ATP) and 2μl of T4 ligase (Fermentas). The ligation was carried out in 18°C overnight (~18h) and then inactivated for 10min. at 65°C.

Results:

Conclusions:

• Despite the low morale we decided we could transform with that.