Construction of the pYPKa vector

The pYPKa is a version of the pCAPs vector where a unique AjiI site has been created by site directed mutagenesis and a AcsI site removed AcsI. The pCAPs plasmid was described by Schlieper et al. 1998 (see below) The abstract is available from Pubmed. The pCAPs sequence is available from Genbank:



In [1]:

    
# NBVAL_IGNORE_OUTPUT
from IPython.display import IFrame
IFrame('http://www.ncbi.nlm.nih.gov/pubmed/9514792', width='100%', height=250)









    Out[1]:

Importing the pydna package. Pydna is open source, documentated here and has a support forum as well as a publication:

Pereira F, Azevedo F, Carvalho Â, Ribeiro GF, Budde MW, Johansson B: Pydna: a simulation and documentation tool for DNA assembly strategies using python. BMC Bioinformatics 2015, 16:142.



In [2]:

    
from pydna.all import *

The plasmid backbone sequence 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]:

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

pCAPS cseguid shoud be S6GoB_Z13VCfGDTEqNclu4It614



In [5]:

    
pCAPs.cseguid()









    Out[5]:





S6GoB_Z13VCfGDTEqNclu4It614

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



In [6]:

    
pCAPs.features = pCAPs.features [1:]

The sequence to be mutated is contained withing the subsequence below:



In [7]:

    
print(str(pCAPs[555:604].seq))









    



TTCTCACTAGTGACCTGCAGCCGGCGCGCCATCTGTGCAGACAAACGCA



In [8]:

    
from Bio.Restriction import AjiI
print(AjiI.site)
print(AjiI.is_palindromic())









    



CACGTC
False

Outline of the mutation strategy

pCAPs sequence

TTCTCACTAGTGACCTGCAGCCGGCGCGCCATCTGTGCAGACAAACGCA   watson  
AAGAGTGATCACTGGACGTCGGCCGCGCGGTAGACACGTCTGTTTGCGT   crick
                   <---ArgAla---

Two aminoacids in the CRP coding sequence are indicated in the crick strand of pCAPs.

Primers annealing to template

TTCTCACTAGTGACCTGCAGCCGGCGCGCCATCTGTGCAGACAAACGCA
  |||||||||||||||||||||
  gagtgatcactggacgtcggcTG
                         GTGCcatctgtgcagacaaacg
                            |||||||||||||||||||
AAGAGTGATCACTGGACGTCGGCCGCGCGGTAGACACGTCTGTTTGCGT

Resulting mutated seqence in pYPKa:

ttctcactagtgacctgcagccgACGTGccatctgtgcagacaaacgca   watson
aagagtgatcactggacgtcggcTGCACggtagacacgtctgtttgcgt   crick
                      ------                        AjiI site

                   <---ArgAla---

The mutation is silent, the codons formed in the CRP orf on the crick strand encode the same aminoacids

      Ala Arg

pCAPS gcg cgg

pYPKa gca cgt



In [9]:

    
primers =parse('''>568_pCAPsAjiIR (22-mer)
                         GTGCcatctgtgcagacaaacg

                         >567_pCAPsAjiIF (23-mer)
                         GTcggctgcaggtcactagtgag''', ds=False)



In [10]:

    
pYPKa =pcr(primers, pCAPs).looped().synced(pCAPs)



In [11]:

    
pYPKa.stamp()









    Out[11]:





cSEGUID_aV1eIrzOiCjvw01yvKkxDXHKLMk



In [12]:

    
pYPKa.name = "pYPKa"



In [13]:

    
pYPKa.write("pYPKa.gb")









    




pYPKa.gb

Download

pYPKa



In [14]:

    
reloaded =read("pYPKa.gb")



In [15]:

    
assert reloaded.cseguid() in reloaded.definition