This notebook describe the construction of E. coli vectors pYPKa_Z_TDH3 and pYPKa_E_TDH3 with the same insert for which PCR primers are also designed.
The insert defined below is cloned in pYPKa using the blunt restriction enzymes ZraI and EcoRV in two different plasmids. The insert cloned in ZraI will be used as a promoter, while in the EcoRV site the insert will be used as a terminator.
The pydna package is imported in the code cell below. There is a publication describing pydna as well as documentation available online. Pydna is developed on Github.
from pydna.readers import read
from pydna.parsers import parse
from pydna.parsers import parse_primers
from pydna.design import primer_design
from pydna.amplify import pcr
from pydna.amplify import Anneal
The vector backbone pYPKa is read from a local file.
pYPKa = read("pYPKa.gb")
Both restriction enzymes are imported from Biopython
from Bio.Restriction import ZraI, EcoRV
The vector is linearized with both enzymes.
pYPKa_ZraI = pYPKa.linearize(ZraI)
pYPKa_EcoRV = pYPKa.linearize(EcoRV)
The insert sequence is read from a local file. This sequence was parsed from the ypkpathway data file.
ins = read("TDH3.gb")
fp_tail = "ttaaat"
rp_tail = "taattaa"
Primers with the tails above are designed in the code cell below.
ins = primer_design(ins)
fp = fp_tail + ins.forward_primer
rp = rp_tail + ins.reverse_primer
The primers are included in the new_primer.txt list and in the end of the pathway notebook file.
print(fp.format("fasta"))
print(rp.format("fasta"))
with open("new_primers.txt", "a+") as f:
f.write(fp.format("fasta"))
f.write(rp.format("fasta"))
>fw698 TDH3 ttaaatATAAAAAACACGCTTTTTC >rv698 TDH3 taattaaTTTGTTTGTTTATGTGTGTTT
PCR to create the insert using the newly designed primers.
prd = pcr(fp, rp, ins)
The PCR product has this length in bp.
len(prd)
711
A figure of the primers annealing on template.
prd.figure()
5ATAAAAAACACGCTTTTTC...AAACACACATAAACAAACAAA3 ||||||||||||||||||||| tm 50.8 (dbd) 56.4 3TTTGTGTGTATTTGTTTGTTTaattaat5 5ttaaatATAAAAAACACGCTTTTTC3 ||||||||||||||||||| tm 48.8 (dbd) 55.3 3TATTTTTTGTGCGAAAAAG...TTTGTGTGTATTTGTTTGTTT5
A suggested PCR program.
prd.program()
Taq (rate 30 nt/s) 35 cycles |711bp 95.0°C |95.0°C | |Tm formula: Biopython Tm_NN |_________|_____ 72.0°C |72.0°C|SaltC 50mM | 03min00s|30s \ ________|______|Primer1C 1.0µM | | \ 50.9°C/ 0min21s| 5min |Primer2C 1.0µM | | \_____/ | |GC 34% | | 30s | |4-12°C
The final vectors are:
pYPKa_Z_TDH3 = (pYPKa_ZraI + prd).looped().synced(pYPKa)
pYPKa_E_TDH3 = (pYPKa_EcoRV + prd).looped().synced(pYPKa)
The final vectors with reverse inserts are created below. These vectors theoretically make up fifty percent of the clones. The PCR strategy below is used to identify the correct clones.
pYPKa_Z_TDH3b = (pYPKa_ZraI + prd.rc()).looped().synced(pYPKa)
pYPKa_E_TDH3b = (pYPKa_EcoRV + prd.rc()).looped().synced(pYPKa)
A combination of standard primers and the newly designed primers are used for the strategy to identify correct clones. Standard primers are listed here.
p = { x.id: x for x in parse_primers("standard_primers.txt") }
The correct structure of pYPKa_Z_TDH3 is confirmed by PCR using standard primers 577 and 342 that are vector specific together with the TDH3fw primer specific for the insert in a multiplex PCR reaction with all three primers present.
Two PCR products are expected if the insert was cloned, the sizes depend on the orientation. If the vector is empty or contains another insert, only one product is formed.
pYPKa_Z_TDH3 with insert in correct orientation.
Anneal( (p['577'], p['342'], fp), pYPKa_Z_TDH3).products
[Amplicon(1645), Amplicon(1477)]
pYPKa_Z_TDH3 with insert in reverse orientation.
Anneal( (p['577'], p['342'], fp), pYPKa_Z_TDH3b).products
[Amplicon(1645), Amplicon(879)]
Empty pYPKa clone.
Anneal( (p['577'], p['342'], fp), pYPKa).products
[Amplicon(934)]
pYPKa_E_TDH3 with insert in correct orientation.
Anneal( (p['577'], p['342'], fp), pYPKa_E_TDH3).products
[Amplicon(1645), Amplicon(1396)]
pYPKa_E_TDH3 with insert in reverse orientation.
Anneal( (p['577'], p['342'], fp), pYPKa_E_TDH3b).products
[Amplicon(1645), Amplicon(960)]
The cseguid checksum for the resulting plasmids are calculated for future reference. The cseguid checksum uniquely identifies a circular double stranded sequence.
print(pYPKa_Z_TDH3.cseguid())
print(pYPKa_E_TDH3.cseguid())
Z8LFAVHm3ruuq_dov31lqpmfeqA 8zg87HoFsdPFl5Ao-Sup64AGvFs
The sequences are named based on the name of the cloned insert.
pYPKa_Z_TDH3.locus = "pYPKa_Z_TDH3"[:16]
pYPKa_E_TDH3.locus = "pYPKa_Z_TDH3"[:16]
Sequences are stamped with the cseguid checksum. This can be used to verify the integrity of the sequence file.
pYPKa_Z_TDH3.stamp()
pYPKa_E_TDH3.stamp()
cSEGUID_8zg87HoFsdPFl5Ao-Sup64AGvFs
Sequences are written to local files.
pYPKa_Z_TDH3.write("pYPKa_Z_TDH3.gb")
pYPKa_E_TDH3.write("pYPKa_E_TDH3.gb")
import pydna
reloaded = read("pYPKa_Z_TDH3.gb")
reloaded.verify_stamp()
cSEGUID_Z8LFAVHm3ruuq_dov31lqpmfeqA
import pydna
reloaded = read("pYPKa_E_TDH3.gb")
reloaded.verify_stamp()
cSEGUID_8zg87HoFsdPFl5Ao-Sup64AGvFs