Construction of the pYPK0 vector

The pYPK0 is a yeast / E. coli shuttle vector version of the pCAPs vector that was made through in-vivo homologous recombination between pCAPs and pMEC1030. The pCAPs vector is described in detail in the notebook describing the pYPKa vector.

Important features are:

A unique ZraI site
A unique EcoRV site
A URA3 marker for selection in S. cerevisiae
The 2µ sequence for replication in S. cerevisiae

The pYPK0 is mainly used as a backbone for the tp_gene_tp cassettes (single gene cassettes) and multi gene pathways. The pYPKpw vector was constructed from the pYPK0.

The strategy used to combine the pMEC1030 and pCAPs vectors into a yeast shuttle vector were described by Iizasa & Nagano 2006.

Briefly, the pMEC1030 vector (pSU0 in the figure above) was digested with BamHI and EcoRI removing a small stuffer fragment. The pCAPs (pBR322 derived vector in the figure above) was digested in the ampicillin marker gene using PvuI (instead of PstI in the figure above). The linear vectors recombine between the ampicillin marker and the pUC origin of replication.



In [1]:

    
from pydna.all import *



In [2]:

    
pMEC1030 = read("pMEC1030.gb")

pMEC1030 (4398 bp)should have cseguid bg67Zek8TFW0rvcIs6-d4X6cV_w



In [3]:

    
print("The pMEC1030 is", len(pMEC1030), "bp long")
print("The cSEGUID checksum is ", pMEC1030.cseguid())









    



The pMEC1030 is 4398 bp long
The cSEGUID checksum is  bg67Zek8TFW0rvcIs6-d4X6cV_w



In [4]:

    
pMEC1030.description









    Out[4]:





'cSEGUID_bg67Zek8TFW0rvcIs6-d4X6cV_w_2018-05-15T07:37:52.355632'

The pCAPs part comes from a Genbank record. Access to Genbank is needed in order to download the template. If you execute this script, change the email address below to your own. Always tell Genbank who you are, when using their web service.



In [5]:

    
gb =Genbank("bjornjobbb@gmail.com")



In [6]:

    
pCAPs = gb.nucleotide("AJ001614.1")

The restriction enzymes needed are imported from Biopython



In [7]:

    
from Bio.Restriction import BamHI, EcoRI, PvuI

The pMEC1030 is cut with BamHI and EcoRI and the pCAPs is cut with PvuI in the amp marker. PvuI was chosen over PstI since in the pCAPs vector PstI cuts in thecrp gene and not in the amp marker.



In [8]:

    
pCAPs.cut(PvuI)









    Out[8]:





(Dseqrecord(-3130),)



In [9]:

    
stuffer, psu_bam_eco = pMEC1030.cut(BamHI, EcoRI)
psu_bam_eco, stuffer









    Out[9]:





(Dseqrecord(-4392), Dseqrecord(-14))



In [10]:

    
pcaps_pvu = pCAPs.linearize(PvuI)



In [11]:

    
psu_bam_eco.name









    Out[11]:





name



In [12]:

    
asm =Assembly((pcaps_pvu, psu_bam_eco), limit=200)



In [13]:

    
candidate = asm.assemble_circular()[0]



In [14]:

    
candidate.figure()









    Out[14]:





 -|AJ001614_lin|700
|               \/
|               /\
|               700|name_rc|245
|                           \/
|                           /\
|                           245-
|                              |
 ------------------------------



In [15]:

    
pYPK0 = candidate.synced(pCAPs)

The cseguid of pYPK0 should be qSJ95NM815X60yBy4l6iI69a_Dw and the size 5762 bp.



In [16]:

    
print(len(pYPK0))
pYPK0.cseguid()









    



5762






    Out[16]:





qSJ95NM815X60yBy4l6iI69a_Dw



In [17]:

    
pYPK0.stamp()









    Out[17]:





cSEGUID_qSJ95NM815X60yBy4l6iI69a_Dw



In [18]:

    
pYPK0.name = "pYKP0"



In [19]:

    
pYPK0.write("pYPK0.gb")









    




pYPK0.gb

Download

pYPK0



In [20]:

    
from pydna.all import *
reloaded =read("pYPK0.gb")
assert reloaded.cseguid() in reloaded.definition