Plasmid_Backbone

Part:BBa_K3425003

Designed by: Núria Garriga Alonso   Group: iGEM20_UofUppsala   (2020-10-19)


pSB3C13: Improved pEven3 Loop Vector based on pSB3C03

The illegal BsaI site was removed from pSB3C03 by PCR mutagenesis to obtain the new pEven3 plasmid pSB3C13.

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

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 pSB3C13 which was obtained from pSB3C03.

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.

Mutagenised Type IIS Level 2 backbones

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.

Detail of the sequencing result of (A) pSB3C01 and (B) pSB3C11

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.

[edit]
Categories
//collections/typeiis/uppsala
//plasmidbackbone/assembly/typeiis
Parameters
insertRFP-device (J04455)
originp15A
resistanceC