Part:BBa_K1639013

pColA-KanR-dTer This part is linear DNA containing ColA1 origin of replication+ Kanamycin resistance+ standard prefix and suffix

Usage and Biology

To express different proteins from different vectors the compatibility issue have to be solved. Because bacteria do not accept vectors with same origins. One example of these compatible origins is ColA-ColE1 origins of replication. Standart expression vectors like pSB1C3/pET-45b(+)/pET-15b(+)/pET-22 all confer origin of ColE1 type. In our project, systems composed of two or more elements were not cloned into one vetor only because of many repeats and high chance of mutation of Taq polymerase. Instead we divided them into two different compatible vectors. (ColA-pET-45b(+)) To achieve this linear version of vector was cirularized with Not1 enzyme (Figure 1) Then gene of interest was cloned into circularized vector with appropriate enzyme in our case Not1. Then postive colonies on Kanamycin plates were selected and incubated in liquid culture to continue with functional assays.

Figure 1: Circularization of linear gBlock

Cloning and Expression

We used this part at cloning and expression step of BBa_K1639000. NotI enzyme was used for cloning and for verification colony PCR was performed by ColA fwd-rev primers (BBa_K1639017-BBa_K1639018). Negative band is expected at 192 bp with these primers. (Figure 2) We tried but we couldn't clone our part into pSB1C3, we think that plasmid containing two origins of replications is lethal to bacteria.

Co-Transformation

To show compatibility of ColA-ColE1 origins below are shown co-transformation experiments conducted for BBa_K1639000. Important point in this experiment is that after cotransformation colonies were screened on Kanamycin+Chloramphenicol plates. Normally the positive GFP expression result is expected at pColA-Toehold GFP + Trigger RNA combination These results prove that our synthetically produced ColA vector is compatible with pSB1C3 carrying ColE origin and also prove that Toehold-Trigger system cotransformed by this part works as expected (Figure 3-4)

Figure 3 Combinations of cotransformations. As expected most flourescence was observed at Toehold GFP+trigger RNA under microscopy

Figure 4 Protein isolation results of combinations.Image taken after centrifugation of bacterial cultures

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found at 1
  Plasmid lacks a suffix.
  Illegal SpeI site found at 1925
  Illegal PstI site found at 1939
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 1
  Illegal NheI site found at 757
  Illegal SpeI site found at 1925
  Illegal PstI site found at 1939
  Illegal NotI site found at 7
  Illegal NotI site found at 1932
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 1
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 1
  Illegal suffix found at 1925
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 1
  Plasmid lacks a suffix.
  Illegal XbaI site found at 16
  Illegal SpeI site found at 1925
  Illegal PstI site found at 1939
  Illegal AgeI site found at 1863
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.