Assembly of endosome detection operon

Marcin

Comment:

Result of following ligations was disappointing (no one bacterial colony on the plates):

• [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]

I examinated the effectiveness of DNA purification. The results reveal that the yield of used gel-out kit was surprisingly low. It forced me to prepare another enzymatic digestions.

Task 1: DNA digest to create following biobricks:

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

Methods:

• Constructs to digest:
  • [http://partsregistry.org/Part:BBa_K177035BBa_K177035] - PstI, SpeI
  • [http://partsregistry.org/Part:BBa_K177035BBa_K177035] - PstI, XbaI
  • [http://partsregistry.org/Part:BBa_K177036BBa_K177036] - PstI, SpeI
  • [http://partsregistry.org/Part:BBa_K177044BBa_K177044] - PstI, XbaI
• Reaction mixture composition:

15 μl purified plasmid DNA product
1 μl enzyme 1 (Fermentas)
1 μl enzyme 2 (Fermentas) 
5 μl Buffer Tango (Fermentas)
28 μl MQ water

• Reactions were carried out 7 hour and subsequently they were inactivated via heating.

PCR of phoP

Monika

Task:

• amplification of phoP (675 bp)

Methods:

• PCR mixture:

 5μl Pfu polymerase buffer
 1μl forward primer and 1μl reverse primer
 2μl dNTPs (10 mM)
 2,5μl Pfu turbo polymerase (EURX)
 2μl template DNA from Salmonella enterica typhimurium LT2
 The solution was topped up with H2O to 50μl
 
 contol - no template DNA from Salmonella enterica typhimurium LT2

• PCR conditions:

   1. 5min 95°C
   2. 30s 95°C
   3. 2min 72°C
   4. go to step 2 x30 
   5. 10min 72°C 
   6. forever 4°C

Results of PCR:

• There were no products of phoP amplification

• There were no products of phoQ amplification

Comment:

• We suppose there were some problems with polymerase and PCR programme was wrongly chosen

Isolation of BBa_J63010 from 2009 Kit

Monika

Tasks:

• Isolate BioBrick [http://partsregistry.org/Part:BBa_J63010 BBa_J63010] from 2009 Kit Plate 1

Methods:

• Alkaline lysis with A&A Biotechnology Kit, procedure described [http://aabiot.com/products/dna_purification/plasmid_dna/plasmid_mini/protocol_plasmid_mini.pdf here]

Monika

Task:

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

Methods:

• PCR mixture:

 5μl Pfu polymerase buffer
 1μl forward primer and 1μl reverse primer
 2μl dNTPs (10 mM)
 2,5μl Pfu turbo polymerase (EURX)
 2μl template DNA from Salmonella enterica typhimurium LT2
 The solution was topped up with H2O to 50μl
 
 
 contol - no template DNA from Salmonella enterica typhimurium LT2

• PCR conditions:

   1. 3min 95°C
   2. 30s 95°C
   3. 35s 59°C
   4. 2min 72°C
   5. go to step 2, 2 times
   6. 30s 95°C
   7. 2min 40s 72°C
   8. go to step 6, 28 times
   9. 10min 72°C 
   10. forever 4°C

Results of PCR:

Cloning of the cro-box into the pSB1A3 plasmid

Kamil

Tasks:

• Selection of the colonies

Methods:

• Additional colonies were selected and transferred to a fresh agarose plate, liquid cultures were established.
• The plate and the liquid cultures were incubated overnight in 37°C