Part:BBa_K2945010

Cut 2 Primer: Primer opposite the Suffix Binding Site

This primer is to be used when creating new BioBricks via Gibson assembly. Currently, creating a new BioBrick requires getting the sequence between the Prefix and Suffix elements. However, these elements can actually anneal to each other, making Biobricks assembly with Gibson using them as overhangs quite tricky. This also means that enzymatic digestion cannot be used to effectively create new BioBricks. This primers site opposite the VF2 primer binding site and points towards the plasmid. This allows it to be used to linearize pSB1C3 so that it has homology with BioBrick-like inserts that contain the VF2 site. This is designed for synthesized BioBricks from either IDT or Twist as these sequences can be added to the desired BioBrick and then can be assembled to pSB1C3 using Gibson Assembly.

This Primer is Compatible for Linearization by PCR with the Cut 1 Primer that is opposite the VF2 Binding Site (BBa_K2945010)

Cut Primer Collection: The Cut Primer Collection (K2945009,K2945010,K2945011, and K2945012) were born out of frustration with Gibson Assembly of new Biobricks. The BioBrick Prefix and Suffix has microhomology to each other, making Gibson Assembly difficult. While trying to assemble our constructs, we encountered several major problems. Many of the inserts we ordered from IDT were using the Prefix and Suffix as our overhang homology to pSB1C3, but many of our transformants from this resulted in plasmids that appeared to have sealed upon themselves rather than taking our insert(Figure 1a), and that the suffix appeared to be missing. We thought it was possible the plasmid was simply religating itself back together due to the overhangs generated by the XbaI and SpeI enzymes , so we treated our digested plasmids with quickCIP (New England Biolabs) which dephosphorylates the 5’ ends and prevents religation. However, we observed the same results as before (Figure 1b).

We then suspected that the Prefix and Suffix may have the capability to anneal to each other and can perform an intramolecular to Gibson reaction rather than taking up our insert. We confirmed this by using <a href="https://www.thermofisher.com/us/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/multiple-primer-analyzer.html">ThermoFisher’s Multiple Primer Analyzer.</a> We chose to do this because the first step of Gibson is to create ssDNA overhangs that can base pair similar to that of primers; if the prefix and suffix did have homology to each other, they appear to create a “primer dimer” at the calculated optimal annealing temperature predicted by the program. Indeed, we found that the Prefix and Suffix do have the capability to anneal to each other with 12 bases of homology to each other in opposite orientation (Figure 2) which would allow for an intramolecular Gibson Assembly to occur instead of our desired insert.

To get around this problem, we developed a series of Cut primers that would allow us to perform Linearization by PCR of pSB1C3. We developed a set of 4 primers, Cut1-Cut4 who sit on either on the VF2, Suffix, Prefix, and VR respectively; This would allow us to use overhangs in common to every iGEM plasmid, allowing an insert to be included in any iGEM vector. Any time we wished to clone an insert into pSB1C3, we would select our primers to linearize pSB1C3 according to Table 1, and put the corresponding overhangs on our insert. This resulted in a nearly 100% efficiency with our Gibson assembly (Figure 3a and 3b). To confirm that this truly was caused by Prefix and Suffix homology, we performed this Gibson with Cut2 and Cut4 which would generate the Linearized plasmid but with both the Prefix and Suffix overhangs. We saw plasmids that had no insert and re-ligated back to itself. We observed our original problem where the plasmid religated back to itself confirming that Prefix and Suffix homology create problems when attempting to use Gibson assembly in the BioBrick Assembly workflow. We hope that our iGEM teams can take advantage of this when trying to assemble new BioBricks into iGEM vectors.

Figure 3A: Gel Showing Colony PCR Gel of an Empty pSB1C3 (Lane 1) and Various Transformed Gibson Products Using Cut Primers that Have Either the Prefix and Suffix (Remaining lanes). NEB 1Kb Plus ladder used

Figure 3B: Gel Showing Colony PCR of pSB1C3 with a 1Kb insert (Lane 1) and Various Transformed Gibson Products Using Cut Primers with Both the Prefix and Suffix Present (Lanes 2-4). NEB 1Kb Plus Ladder used

Table 1: Compatibility of primers in the Cut Series

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal suffix found in sequence at 1
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal suffix found in sequence at 2
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  
• 1000
  COMPATIBLE WITH RFC[1000]