Construction of the pMEC1030 vector

The pMEC1030 is a version of the pSU0 vector where the single EcoRV site has been removed by site directed mutagenesis. The pSU0 plasmid was described in by Iizasa Nagano 2006. The abstract is available from Pubmed. The pSU0 sequence is available from Genbank.

The pSU0 plasmid has the following structure



In [1]:

    
# NBVAL_IGNORE_OUTPUT
from IPython.display import IFrame
IFrame('http://www.ncbi.nlm.nih.gov/pubmed?LinkName=nuccore_pubmed&from_uid=84570488', width='100%', height=250)









    Out[1]:



In [2]:

    
from pydna.all import *

The insert 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 the service.



In [3]:

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



In [4]:

    
pSU0 = gb.nucleotide("AB215109.1")

The first feature is removed for practical reasons since it covers the whole sequence.



In [5]:

    
pSU0.features = pSU0.features[1:]

pSU0 is a circular DNA molecule with 4398 bp. The cseguid checksum should be fzXVMVR4-lIsvU_eXXMG10U18s4.



In [6]:

    
pSU0.cseguid()









    Out[6]:





fzXVMVR4-lIsvU_eXXMG10U18s4



In [7]:

    
pSU0









    Out[7]:




AB215109.1



In [8]:

    
from Bio.Restriction import EcoRV

The pSU0 has one EcoRV site which we would like to remove, since the Yeast Pathway Kit relies on unique EcoRV, AjiI and ZraI sites. One linear fragment is formed when digested by EcoRV:



In [9]:

    
pSU0.cut(EcoRV)









    Out[9]:





(Dseqrecord(-4398),)

Two primers were designed for the specific removal of EcoRV through PCR amplification and susequent yeast transformation of the linear PCR product. The PCR product has two 50 bp intramolecular repeats at both ends of the molcule which are repaired into a circular molecule with the same size as the original vector.



In [10]:

    
primers =parse('''
>470_pSU0f-dEcoRV (50-mer)
gtttactaaaaacacatgtggatattttgactgatttttccatggagggc

>469_pSU0r-dEcoRV (50-mer)
gccctccatggaaaaatcagtcaaaatatccacatgtgtttttagtaaac''', ds=False)



In [11]:

    
pSU0_EcoRV =pcr(primers, pSU0)

The figure below show the location of the primers on the circular template.



In [12]:

    
pSU0_EcoRV.figure()









    Out[12]:





                          5ttgactgatttttccatggagggc...gtttactaaaaacacatgtggatat3
                                                      ||||||||||||||||||||||||| tm 53.1 (dbd) 58.2
                                                     3caaatgatttttgtgtacacctataaaactgactaaaaaggtacctcccg5
5gtttactaaaaacacatgtggatattttgactgatttttccatggagggc3
                           |||||||||||||||||||||||| tm 60.8 (dbd) 71.8
                          3aactgactaaaaaggtacctcccg...caaatgatttttgtgtacacctata5

The DNA is processed in vivo to produce 5' cohesive ends.



In [13]:

    
from pydna.dseq import Dseq
pSU0_EcoRV_proc =Dseqrecord(Dseq(pSU0_EcoRV.seq.watson[50:], pSU0_EcoRV.seq.crick[50:]))

The figure below shows the cohesive ends.



In [14]:

    
pSU0_EcoRV_proc.seq









    Out[14]:





Dseq(-4448)
          ACAG..ATTTGTTT..gggc
caaa..CCCGTGTC..TAAA

The plasmid is cirularized in-vivo after transformation to yeast.



In [15]:

    
pMEC1030 = pSU0_EcoRV_proc.looped().synced("ggatccatcggaattcatattgaaaaagga")

The pMEC1030 has no EcoRV site as expected.



In [16]:

    
pMEC1030.cut(EcoRV)









    Out[16]:





()



In [17]:

    
pMEC1030.add_feature(2741,3545, label= "URA3")



In [18]:

    
pMEC1030.stamp()









    Out[18]:





cSEGUID_bg67Zek8TFW0rvcIs6-d4X6cV_w



In [19]:

    
pMEC1030.add_feature(3625,4307, label = "ColE1 origin", type="rep_origin")
pMEC1030.add_feature(234, 893, label = "AmpR", type="CDS")



In [20]:

    
pMEC1030.name = "pMEC1030"



In [21]:

    
pMEC1030.write("pMEC1030.gb")









    




pMEC1030.gb



In [22]:

    
from pydna.all import *
reloaded =read("pMEC1030.gb")
assert reloaded.cseguid() in reloaded.definition