Part:BBa_K3425002

pSB3C12: Improved pEven2 Loop Vector based on pSB3C02

The illegal BsaI site was removed from pSB3C02 by PCR mutagenesis to obtain the new pEven2 plasmid pSB3C12.

iGEM Type IIS Vectors

pEven Loop Vectors (for Level 2)

When four Level 1 parts (TUs) are assembled into a Multi-Transcriptional Unit (MTU) into the following plasmid backbones (with BBa_J04455), the MTU will be flanked by BsaI restriction sites and these 4 bp Fusion Sites.

Part number	Name	Loop Name	Fusion Site 5'	MTU	Fusion Site 3'
BBa_K3425001	pSB3C11	pEven1	GGAG	MTU 1	TACT
BBa_K3425002	pSB3C12	pEven2	TACT	MTU 2	AATG
BBa_K3425003	pSB3C13	pEven3	AATG	MTU 3	GCTT
BBa_K3425004	pSB3C14	pEven4	GCTT	MTU 4	CGCT

The iGEM Type IIS assembly standard is based on MoClo and Loop

• A Modular Cloning System for Standardized Assembly of Multigene Constructs
• Loop assembly: a simple and open system for recursive fabrication of DNA circuits

Note: This section was adapted from pSB3C01 in order to collect all the information in one page. More information, as well as all our experience in this cloning method, can be found in Team Uppsala 2020's iGEM Type IIS standard guidebook.

This part is part of iGEM Uppsala 2020's iGEM Type IIS standard collection.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2713
  Illegal PstI site found at 12
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2713
  Illegal NheI site found at 1399
  Illegal PstI site found at 12
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2713
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2713
  Illegal PstI site found at 12
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 2713
  Illegal PstI site found at 12
  Illegal AgeI site found at 990
  Illegal AgeI site found at 1313
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal BsaI site found at 2719
  Illegal BsaI.rc site found at 6
  

Mutagenesis of pSB3C0# pEven Loop vectors

The sequence of pEven Loop vectors for Level 2 assemblies (pSB1C0#) contains an additional, illegal BsaI site. BsaI is the enzyme which is used to cleave the assembly out of a pEven vector to be cloned back to a pOdd vector. Therefore, the presence of this illegal site would impact the construction of Level 3 assemblies. Site-directed mutagenesis by PCR was conducted in order to remove the illegal sites. The new plasmids were named pSB3C1#. This page corresponds to pSB3C12 which was obtained from pSB3C02.

After PCR mutagenesis, screening was done by digestion of the plasmid DNA with BsaI (Figure 1). All backbones (pSB3C0# and pSB3C1#) contain a band that corresponds to the mRFP1 cassette (BBa_J04455) with a size of 1093bp. Then, pSB3C1# consist of one more band of 2725pb. Since pSB3C0# have one more BsaI site, they should present two more bands of 2434bp and 291bp. While the 291bp band is not visible, probably due to too low DNA amount, the shift in size before and after mutagenesis is clearly visible.

Figure 1. 1% Agarose Gel Electrophoresis - SYBR Safe analysis of BsaI digested plasmids. MW marker is GeneRuler 1kb Plus DNA Ladder (Thermofisher). pSB3C03 was linearised with EcoRI.

This difference cannot be seen, however, for pSB3C01 and pSB3C11. In addition, a longer band can be seen for pSB3C01, but a comparison to the linearised pSB3C03 shows that it corresponds to linearised plasmid. To investigate the lack of difference between pSB3C01 and pSB3C11, as well as to confirm the mutagenesis by another method, pSB3C01 and pSB3C1# were sequenced.

Figure 2. Detail of the sequencing result of (A) pSB3C01 and (B) pSB3C11. At the upper part, the BsaI cutting site and mutagenesis primers are shown. At the bottom are the corresponding sequencing reads.

Sequencing results showed a successful PCR mutagenesis for all of the pSB3C1# backbones, with the expected point mutation (Figure 2, B). Furthermore, they also showed a point mutation in the original backbone, pSB3C01, (at a different point than the planned one) which eliminated the extra BsaI site (Figure 2, A).

Afterwards, some assemblies into pSB3C1# were conducted, which show their functionality. One such example is part BBa_K3425072. This confirmed that these backbones would work as the new pEven set of vectors.

This information can also be found in Team Uppsala 2020's Wet lab page. Sequencing results of this part with J04455 can be found in the Sequence Analysis section and the trace files are in the collection page as well as our Parts page.