Assembly of endosome detection operon

Marcin

Task 1: Prepare the following ligations:

• [http://partsregistry.org/Part:BBa_K177035BBa_K177035] with [http://partsregistry.org/Part:BBa_K177036BBa_K177036] to obtain [http://partsregistry.org/Part:BBa_K177037BBa_K177037]
• [http://partsregistry.org/Part:BBa_K177035BBa_K177035] with [http://partsregistry.org/Part:BBa_K177043BBa_K177043] to obtain [http://partsregistry.org/Part:BBa_K177044BBa_K177044]

Methods:

• Reaction mixtures composition:

45 μl mixture of insert and vector DNA (both are purified at the same probe)
1.5 μl T4 ligase (Fermentas)
5 μl Ligase Buffer (Fermentas)

• Ligation was carried out 6 hours in 16 °C. Subsequently both mixtures were divided up two portions. In the case of each reaction one portion was thermally inactivated and the latter one was unremoved.

Task 2: Transformation of TOP10 chemocompetent bacteria with following constructs:

• [http://partsregistry.org/Part:BBa_K177044BBa_K177044]
• [http://partsregistry.org/Part:BBa_K177037BBa_K177037]

Methods:

• Ligation mixture was thermally inactivated
• Detailed protocol of transformation is described here

PCR of phoP and phoQ

Monika

Task:

• amplification of phoP (675 bp) and phoQ (1464 bp)

Methods:

• PCR mixture:

 1μl Pfu polymerase buffer
 0,2μl forward primer and 1μl reverse primer
 0,4μl dNTPs (10 mM)
 0,5μl Pfu turbo polymerase (EURX)
 0,4μl template DNA from Salmonella enterica typhimurium LT2
 The solution was topped up with H2O to 10μl
 
 contol - no template DNA from Salmonella enterica typhimurium LT2

• PCR conditions:

   1. 3min 95°C
   2. 30s 95°C
   3. 35s 58°C
   4. 2min 10 s 72°C
   5. go to step 2, 2 times
   6. 30s 95°C
   7. 30s 68°C
   8. 2min 10s 72°C
   9. go to step 6, 28 times
   10. 10min 72°C 
   11. forever 4°C

Results of PCR:

Bistable switch testing

Task: Control digestion of bistable switch

Methods

• Reaction mixture composition:

     6μl plasmid solution
     0,3μl HindIII (Fermentas)
     0,3μl NheI (Fermentas) 
     2μl Buffer Tango (Fermentas)
     11,4 μl MQ water

• Digestion in 37°C for 2 h

Results

• Will be seen tomorrow

Cloning of the cro-box into the pSB1A3 plasmid

Kamil

Tasks:

• Cro-box preparation
• Ligation

Methods:

• Two short oligonucleotides were annealed to form a complete double stranded cro-box sequence with XbaI and PstI sticky ends. Equal concentrations of both oligonucleotides were mixed together, heated to 95°C for 15 min. and left to cool.
• The pSB1A3 plasmid was digested with XbaI and PstI and the resulting backbone was isolated (all as described previously).
• The ligation was carried out in 18°C for 4h.