Difference between revisions of "Part:pSB1K04"

m
(→‎Uppsala 2020's Addition)
 
(2 intermediate revisions by 2 users not shown)
Line 6: Line 6:
  
  
−
===iGEM TypeIIS Vectors===
 
−
====pOdd Loop Vectors (for Level 1)====
 
 
{{CSS/DNA_Table}}
 
{{CSS/DNA_Table}}
 
{{TypeIIS/Plasmids/Level1}}
 
{{TypeIIS/Plasmids/Level1}}
Line 27: Line 25:
 
<partinfo>pSB1K04 parameters</partinfo>
 
<partinfo>pSB1K04 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
 +
==Uppsala 2020's Addition==
 +
<html>
 +
<p>Since high copy number plasmids are not suitable for higher level assemblies, we have assembled 3 sets of new pOdd plasmids for different situations:</p>
 +
 +
<ul>
 +
<li>pSB3K0# (</html><partinfo>BBa_K3425005</partinfo><html> to </html><partinfo>BBa_K3425008</partinfo><html>): medium copy number (10-12 copies)</li>
 +
<li>pSB4K0# (</html><partinfo>BBa_K3425009</partinfo><html> to </html><partinfo>BBa_K3425012</partinfo><html>): low copy number (~5 copies) </li>
 +
<li>pSB4A0# (</html><partinfo>BBa_K3425013</partinfo><html> to </html><partinfo>BBa_K3425016</partinfo><html>): low copy number (~5 copies) with different resistance for cotransformations</li>
 +
</ul>
 +
</html>
 +
 +
==TU_Dresden 2022's Addition==
 +
To make a plasmid suitable for yeast, we made several modification to pOdd vector, as well as added auxotrophy selective markers:
 +
*URA3 marker [https://parts.igem.org/Part:BBa_K4365022 BBa_K4365022]
 +
*HIS3 marker [https://parts.igem.org/Part:BBa_K4365024 BBa_K4365024]
 +
*TRP1 marker [https://parts.igem.org/Part:BBa_K4365023 BBa_K4365023]

Latest revision as of 20:46, 13 October 2022


pOdd4 Loop Vector based on pSB1K3

pOdd4 Loop Vector based on pSB1K3


Assembling into Level 1 Plasmds

pSB1K01-pSB1K04 series of plasmids are based on the pOdd set of Loop Vectors and are built upon the pSB1K3 plasmid backbone.

Note:

  • Since pSB1K3 is a high-copy plasmid backbone, these vectors should not be used for Level 3 assemblies.
  • Currently, Registry samples of these plasmid backbones have not been fully sequenced. Only their prefix and suffix along with their insert BBa_J04454 have been verified.


TypeIIS-Level1-J04454-Simple.png
Simplified diagram of BBa_J04454 and pSB1K0# with BsaI cut site.


When four Level 0 parts are assembled into the plasmid backbone (paired with BBa_J04454) in a one-pot assembly with BsaI...

TypeIIS-Level1-L0-Part-Assembly-Simple.png


the resulting transcriptional unit (TU) will be flanked by SapI restriction sites and the specified 3 bp Fusion Sites (NNN).

TypeIIS-Level1-TU-Simple.png



Level 1 Plasmid Set

Level 1 transcriptional units in this set of plasmid backbones will be flanked by SapI recognition sites and specified 3bp fusion sites, enabling further assembly of up to 4 transcriptional units as a Level 2 Assembly.

Registry Name Loop Name Fusion Site 5' TU Fusion Site 3'
pSB1K01 pOdd1 ATG TU 1 GCA
pSB1K02 pOdd2 GCA TU 2 TAC
pSB1K03 pOdd3 TAC TU 3 CAG
pSB1K04 pOdd4 CAG TU 4 GGT

Level 1 -> Level 2 Assembly

Up to 4 Level 1 transcriptional units can be assembled into a Level 2 multi-transcriptional unit. By planning your assemb

TypeIIS-Level1-Plasmid-Set.png
Simplified diagram of pSB1K0# set with assembled transcriptional units (TUs).


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


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 2179
    Illegal PstI site found at 12
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 2179
    Illegal PstI site found at 12
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 2179
    Illegal XhoI site found at 1029
    Illegal XhoI site found at 2055
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 2179
    Illegal PstI site found at 12
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 2179
    Illegal PstI site found at 12
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 2185
    Illegal SapI.rc site found at 5


Uppsala 2020's Addition

Since high copy number plasmids are not suitable for higher level assemblies, we have assembled 3 sets of new pOdd plasmids for different situations:

TU_Dresden 2022's Addition

To make a plasmid suitable for yeast, we made several modification to pOdd vector, as well as added auxotrophy selective markers: