Part:BBa_K3781105:Design

weird_plex, MocloMania expression vector

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1689
  Illegal EcoRI site found at 2487
  Illegal XbaI site found at 2514
  Illegal SpeI site found at 5191
  Illegal PstI site found at 2526
  Illegal PstI site found at 3508
  Illegal PstI site found at 4831
  Illegal PstI site found at 5822
  Illegal PstI site found at 6579
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1689
  Illegal EcoRI site found at 2487
  Illegal NheI site found at 1008
  Illegal SpeI site found at 5191
  Illegal PstI site found at 2526
  Illegal PstI site found at 3508
  Illegal PstI site found at 4831
  Illegal PstI site found at 5822
  Illegal PstI site found at 6579
  Illegal NotI site found at 2737
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1689
  Illegal EcoRI site found at 2487
  Illegal BglII site found at 2130
  Illegal BamHI site found at 2508
  Illegal BamHI site found at 4085
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1689
  Illegal EcoRI site found at 2487
  Illegal XbaI site found at 2514
  Illegal SpeI site found at 5191
  Illegal PstI site found at 2526
  Illegal PstI site found at 3508
  Illegal PstI site found at 4831
  Illegal PstI site found at 5822
  Illegal PstI site found at 6579
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1689
  Illegal EcoRI site found at 2487
  Illegal XbaI site found at 2514
  Illegal SpeI site found at 5191
  Illegal PstI site found at 2526
  Illegal PstI site found at 3508
  Illegal PstI site found at 4831
  Illegal PstI site found at 5822
  Illegal PstI site found at 6579
  Illegal NgoMIV site found at 1675
  Illegal NgoMIV site found at 4368
  Illegal NgoMIV site found at 4429
  Illegal NgoMIV site found at 5364
  Illegal NgoMIV site found at 5598
  Illegal NgoMIV site found at 5669
  Illegal NgoMIV site found at 6734
  Illegal AgeI site found at 5412
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2726
  Illegal BsaI.rc site found at 2142
  Illegal SapI site found at 2090
  

Design Notes

The domestication of this destination vector was by far the most time-consuming step to generating our MoClo library. The initial pLEXSY_I-blecherry3 template had already been purchased from Jena Bioscience by our supervising working group due to their experimental work with Leishmania tarentolae. After transforming and replicating the original plasmid in TOP10 E. Coli, the domestication of three endogenous BsaI restriction sites was supposed to be conducted via a simple PCR. Specially designed primers would introduce a silent, single nucleotide mutation eliminating the restriction sites and separating the plasmid into three PCR fragments. By introducing additional, terminal BsaI restriction sites at the very ends of the primers, the PCR fragments should easily recombine into a whole plasmid via a MoClo ligation.

While two out of three PCRs worked out on the first try, one fragment of the plasmid was not once successfully and entirely amplified by PCR. Sequencing of the troublesome fragment always showed a particular region within the pLEXSY_I-blecherry3 plasmid to be missing bases. This region, labelled FU by our team, was extremely GC rich and contained a sequence of 16 consecutive Cs. This was more than our Q5 polymerase was able to handle, so we had to come up with a different approach.

The second idea was to assemble the domesticated vector with the missing basepair fragment and then clone the original region from pLEXSY_I-blecherry3 into its domesticated version. But since we had to be careful not to re-introduce any endogenous BsaI restriction sites, only a very limited set of restriction enzymes was able to do the job. The only one to cut upstream of the FU region and downstream of an endogenous BsaI site was ClaI, an enzyme whose restriction activity is blocked by Dam methylation. Since our pLEXSY_I-blecherry3 as well as our assembled vector were both very much Dam methylated, we had to find a Dam- E. Coli strain and transform the plasmids. Thanks to our microbiology department, we were able to get our hands onto the CGSC5127 Dam- E. Coli strain soon and, after a lot of trial and error, were able to transform and prep our non-methylated vectors successfully. After this, a classic cloning procedure ensued, cutting the troublesome region out of the original pLEXSY_I-blecherry3 and introducing it into our domesticated version. Finally, this was successful and we were able to verify our new weird_plex via sequencing.

To learn more about this design process, please feel free to check out our wiki for more information!

Source

The plasmid's sequence was designed and constructed by the scientific staff at Jena Bioscience. The sequences included within the plasmid are either derived from E. Coli (origin of replication), Aequoria victoria (mCherry), E. Coli phages (T7 system), other microbial organisms or synthetic sources (ampicillin/bleomycin resistance). Please consult Jena Bioscience for more detailed information.