Assembly of invasion operon

Marcin

Task 1:

• Prepare chemocompetent bacteria

Detailed protocol

1. Incubate 100 ml of liquid bacterial culture until the OD value is 0.55 or little more
2. Incubate the bacteria on the ice for 15 minutes
3. Centrifuge the bacteria at 4°C for 15 minutes
4. Remove the supernatant and add 10 ml of 0.1 M solution of CaCl₂
5. Gently suspend the bacterial pellet and incubate the bacteria on the ice for 45 minutes
6. Centrifuge the bacteria at 4°C for 15 minutes
7. Remove the supernatant and add 2 to 5 ml of 0.1 M solution of CaCl₂
8. Prepate the aliquots of suspended bacteria which contain 100 μl of the solution
9. Store the bacteria in -80°C

Assembly of endosomal detection operon

Marcin

Task 1:

• Transformation of chemocompetent E. coli strain DH5α

Methods:

• Detailed protocol of transformation is described here. The only modification is usage of total volume of ligation mixture prepared 20.07.09
• petri dish were hold in 37°C for 16 hours

Cloning of p53 coding sequence

Marcin

Task 1:

• Cloning p53 coding sequence to pKS plasmid

Methods:

• Ligation mixture composition: 10 μl digested p53, 25 μl digested pKS, 5 μl ligation buffer (Fermentas),8 μl MQ water, 2 μl ligase T4
• Duration of ligation was about 7 hours; reaction was conducted in 16 °c (approximately).

Task 2:

• Transformation of chemocompetent E. coli strain DH5α

Methods:

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

Parts preparations

Sebastian

Task 1:

• Digest of parts BBa_B0032, BBa_C0040, BBa_C0051, BBa_E0032

Methods:

• Digest of BBa_C0040, BBa_C0051 and BBa_E0032 with PstI/XbaI and BBa_B0032 with PstI/SpeI
• Reaction mixture (BBa_C0040, BBa_C0051 and BBa_E0032): 10ul of plasmid, 1ul of each enzyme, 5ul of Fermentas Tango Yellow buffer, 33ul H2O
• Reaction mixture (BBa_B0032): 5ul of plasmid, 0,5ul of each enzyme, 2,5ul of Fermentas Tango Yellow buffer, 16ul H2O
• Time of digest - 6h

Task 2:

• Electrophoresis and gel-out

Methods:

• Electrophoresis of all samples volume
https://static.igem.org/mediawiki/2009/a/ab/Trawienia22-07.JPG
• Order of samples: (1,2) BBa_E0032, (3,4) BBa_C0051, (5) generuler DNA Mix, (6,8,9) BBa_C0040, (7) BBa_B0032
• Gel-out of samples with A&A Biotechnology Kit
• Electrophoresis of the samples after Gel-out
https://static.igem.org/mediawiki/2009/8/8a/Gel-out.JPG
• Order of samples: BBa_B0032, BBa_C0040, BBa_C0051, BBa_E0032, generuler DNA mix

Cloning of the mgtc promoter into the pKSII+ plasmid

Kamil

Tasks:

• Transformation verification

Methods:

• In order to save on resources colonies that potentially contain the mgtc promoter (the white ones) were marked and the entire dish returned to 37°C for the next 24h.

Construction of K177012 operon1_part2

Ania

Tasks:

• Gel extraction of BBa_B0032- RBS.3 on the pSB1A2 ampicillin resistant plasmid and gel extracion of - lacI repressor
• Overnight ligation of extracted fragments