Generation of pKANUC Recombinant DNA from pUC & pKAN Plasmids
Department of Biological Sciences, Florida Institute of Technology
By Marilla Andrews, Julio Francisco, Esjoy Moreno, Alyson Vezina, and Shukun Yang
Abstract
The purpose of this experiment
was to insert the kanamycin
resistance gene (Kanr) into
pUC19 to produce recombinant
plasmids. To achieve this,
digestion of pUC19 and pKAN
was performed by restriction
enzymes, BamHI and HindIII,
which allowed for the
recombination of the plasmids
by DNA ligase. Recombinant
plasmids were identified by
their ability to survive in the
presence of ampicillin (AMP)
and kanamycin (KAN).
Background
In bacterial transformation
systems, two main components
are required: a suitable host
bacteria and a plasmid. In order
to produce a recombinant
molecule, the vector and insert
DNA are cut with compatible
restriction enzymes allowing
DNA recombination by DNA
ligase. Uptake of the plasmid
by host cells may be induced
by variety of techniques,
increasing permeability to DNA.
Recombinant colonies are
identified via screening for
antibiotic resistance or
colorimetric indications.
Recombinant transformation of
host cells is an effective method
to modify DNA and therefore
change the properties of the
recipient lending itself to a
variety of applications from
agriculture to medicine (1-3).
Materials & Methods Conclusion
Isolated pUC19 and pKAN DNA were double digested using BamHI and HindIII. In conclusion, the E. coli
Recombination of the digested DNA was performed with an experimental 1:8 DH5 host cells were
(vector/insert) ligation ratio. CaCl 2 treated E.coli DH5 were transformed with the rDNA, successfully transformed
and plated on LB-AMP plates. Screening of the using the pKANUC
experimental ligation was done on LB-AMP, LB- recombinant plasmid DNA.
KAN, and LB-AMP/ X-gal/ IPTG media to select The analysis of the isolated
transformed host cells with the rDNA. The rDNA DNA from the E. coli DH5
was isolated and analyzed via agarose gel host cells transformed with
electrophoresis. Why Gas-phase?
pUC19 and pKAN (after
being subjected to the double
digest) showed the
appropriate banding patterns
that corresponds to the AmpR
and KanR gene from pUC19
and pKAN. (Fig 1)
The screening indicated
seven pKANUC transformed
Figure 2. Construction and ligation of
Figure 1. Transformed E. coli DH5 cells and Isolated pUC19 & pKAN Plasmids to produce
colonies exhibiting AMPR,
plasmid double digest on agarose gel (B). the pKANUC plasmid. KANR, and white colony
formation on LB-AMP, X-gal,
Results IPTG media. (Fig 3)
Table 1. Screening of Recombinant Transformed The agarose gel analysis of
Cells for Experimental 1:8 Ligation Ratio. + the plasmid isolation of the
indicates growth.,- indicates no growth, W pKANUC recombinant
indicates white color, and B for blue color. Uses host
transformed cells
supports the indication that
pKANUC was produced.
(Fig 4)
This particular experiment
Figure 3. Antibiotic and Colorimetric Screening for provided some clarity about
the Recombinant bacterias ability to intake
genetic material and use it for
its benefit.
References
1.Johnson, I. (1983) Human insulin
from recombinant DNA
technology. Science 219.
2.Polson, C. D., (2015). Lab Manual
G.E. Techniques.
3.Villarejo M., Langley K., & Fowler,
(1975) Molecular basis of
Figure 4. Agarose Gel Electrophoresis Analysis. -HindIII DNA marker (lane 1), pUC19 (lane 2), beta-glactosidase alpha-
pKAN (lane 3), and recombinant DNA (lane 4). All DNA samples were digested with HindIII and complementation PNAS. 72,
1254-1257.
BamHI. Red lines indicate banding patterns that corresponds to the Amp R and KanR gene.