Eff icient and Easy Vector Modification - www.genscript.com
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
www.genscript.com Efficient and Easy Vector Modification Using GenParts™ DNA Fragments Application Note
www.genscript.com
Efficient and Easy Vector Modification Using
GenParts™ DNA Fragments
Abstract
Modifying vectors to suit the specific objectives of each experiment is key in molecular biology. Yet, the
conventional vector modification technique through primer design and PCR is tedious, prone to errors and when
working with de novo sequences, not feasible. In this application note, we discuss how double-stranded GenParts™
DNA fragments can efficiently and reliably be used to insert desired sequences in a vector.
Introduction
As self-replicating extrachromosomal circular DNAs, vectors are considered essential tools in molecular biology (1).
Their versatility in carrying different sequences allows them to be used for gene amplification, expression,
manipulation and detection in desired host organisms (2). Traditionally, PCR amplification has been used to modify,
insert or replace parts, such as promoter regions, open reading frames or tags, in a vector to obtain the desired vector
(3-4). Also, access to commercial vectors have made it easy for researchers to choose a specific vector containing
their desired features.
There are, however, instances where neither PCR nor commercial vectors can provide an ideal vector and
researchers have to rely on in house vector modification. For example, when the experimental objective is to test the
expression of a de novo sequence, PCR amplification or commercial vectors cannot provide the desired construct
because there is no template available: the de novo sequence has to be synthetically made and then inserted into a
vector. Also, in cases where a highly-customized construct need to be slightly modified for expression or detection in
a new host organism or with a new reporter tag, in house modification provides the fastest, most reliable and
cost-efficient approach: PCR amplification or rounds of modification and parts transfer using commercial vectors can
increase error rate, timeline and cost.
Now, with access to advanced gene synthesis technologies, an alternative approach is available for easy and
efficient in house vector modification. Synthetic double-stranded DNA sequences, called DNA fragments or blocks,
can be chemically synthesized to contain either natural, modified or de novo sequences corresponding to any part
in a vector. GenParts™, custom DNA fragments synthesized by GenScript, are synthesized according to a
custom-designed sequence in an industrial setting with high fidelity and without a template. These fragments,
ranging from 100-2000 bps in length, can then be cloned into any vector of choice using any cloning method or
assembly kit. Application of GenParts™ DNA fragments for vector modification provides experimental feasibility and
savings in time, money and effort towards enabling and expediting novel research projects.
860 Centennial Ave., Piscataway, NJ 08854, USA
Toll-Free: 1-877-436-7274 | Tel: 1-732-885-9188 | Fax: 1-732-210-0262 | Email: info@genscript.com 1
www.genscript.com
Objective and Approach
To demonstrate the advantages of using GenParts™ DNA fragments for vector modification, we added three parts in
a commonly-used vector, pET28a, namely pBAD promoter, GFP tag and AmpR marker (Fig. 1, Table 1). First,
GenParts™ DNA fragments were designed for optimal expression combined with homologous sequences at their 3’
and 5’ ends for subsequent isothermal cloning. After controlling the quality of synthesized fragments, GenParts™
fragments and linearized pET28a vector were gel extracted, purified and cloned using the GenBuilder™ Plus
Cloning Kit (Cat. #L00744, GenScript; Fig. 2). Transformed Top10 competent E. coli cells were then screened on
selective media. To check the accuracy and efficiency of vector modifications, sixteen colonies from control (i.e.
bacteria transformed with unmodified vector) and experimental (i.e. bacteria transformed with modified vectors)
plates were subjected to colony PCR (Fig. 3) followed by sequencing. Fluorescence imaging and growth on
selective media were also performed for GFP- and AmpR-transformed cells, respectively. For detailed protocol, visit
our GenParts™ Resource Center.
moter
BAD pro
or p
art f
nP
Ge
Ge
nP
art
f1 or
i
for
00
50 MCS T7 terminator
Am
is
P
6xH
r GF
p
GenPart fo
Ka
nR
pET28a
LicI
5369 bp
4000
i
or
00
20
3000 rop
Figure 1. Map of pET28a vector indicating target and replaced parts.
860 Centennial Ave., Piscataway, NJ 08854, USA
Toll-Free: 1-877-436-7274 | Tel: 1-732-885-9188 | Fax: 1-732-210-0262 | Email: info@genscript.com 2
www.genscript.com
pET28a-pBAD
1292 bp
GenParts pET28a pET28a-GFP
793 bp
pET28a-Amp
931 bp
Figure 2. Gel electrophoresis of linearized vectors and Figure 3. PCR results of transformed clones.
GenPartsTM DNA fragments. (M: DNA ladder)
Results
Sequencing results from six PCR-positive clones in each vector-modified plate, showed 100% efficiency in
replacement and orientation of fragments within the modified pET28a vector (Table 1). Results from fluorescence
imaging of GFP-expressing bacteria as well as growth of AmpR-containing bacteria on selective media, further
confirmed the successful modification of pET28a with GenParts™ DNA fragments (Fig. 4-5).
Length of # Selected Clones # Selected Clones
Target Part Replaced Part # PCR-Positive Clones # Correct Clones
Fragmen for PCR Testing for Sequencing
T7 Promoter pBAD Promoter 1292 16 14 6 6
T7 Tag GFP Tag 793 16 11 6 6
Kan Marker
R
Amp Marker
R
932 16 16 6 6
Table 1. Modifications made in pET28a using GenPartsTM and corresponding cloning results.
860 Centennial Ave., Piscataway, NJ 08854, USA
Toll-Free: 1-877-436-7274 | Tel: 1-732-885-9188 | Fax: 1-732-210-0262 | Email: info@genscript.com 3
www.genscript.com
pET28a-pBAD pET28a-GFP pET28a-Amp on Amp+ plate pET28a-Amp on Kan+ plate
Figure 4. Growth of bacteria containing modified pET28a vector on selective media.
Control Unmodified pET28a-Transformed Bacteria pET28a-GFP-Transformed Bacteria
Figure 5. Fluorescence imaging of bacteria containing pET28a vector with or without GFP tag.
Conclusion
Sequence-verified GenParts™ DNA fragments are superior alternatives to PCR-obtained sequences for vector
modification. With high sequence accuracy and versatility in design, GenParts™ DNA fragments allow you to (a)
easily modify any vector without the hassle of primer design and PCR optimization to obtain reliable results while
saving time, cost, and effort, and (b) work with any de novo sequence for successfully executing novel research
projects.
860 Centennial Ave., Piscataway, NJ 08854, USA
Toll-Free: 1-877-436-7274 | Tel: 1-732-885-9188 | Fax: 1-732-210-0262 | Email: info@genscript.com 4
www.genscript.com
Advantages of GenParts™ DNA Fragments
Generate natural or synthetic sequences without a template
Assemble fragments into larger DNA blocks
Avoid primer design and PCR optimization
Get your DNA in half the time required for standard gene synthesis
References
1. Sambrook J., Russell DW. Molecular cloning: a laboratory manual. Cold Spring Harbor; New York: 2001.
2. Cohen SN. Bacterial plasmids: their extraordinary contribution to molecular genetics. Gene. 1993; 135:67–76.
3. Innis MA. et al. . PCR protocol: A guide to methods and applications. Academic Press; San Diego: 1990.
4. Erlich HA. et al. Recent advances in the polymerase chain reaction. Science. 1991; 252:1643–1651.
5. Rodrigues RT., Bayer TS. One-Step Isothermal Assembly of DNA fragments. Methods Mol Biol. 2013; 1073:43-7.
Advantages of GenBuilder™ Cloning Kits
Works with unpurified PCR fragments
The most efficient kit among eight competing DNA assembly kits
Allows successful assembly of 12 DNA fragments with >90% positive rate
Performance-verified by >1,000 reactions/day in customers’ assembly projects
860 Centennial Ave., Piscataway, NJ 08854, USA
Toll-Free: 1-877-436-7274 | Tel: 1-732-885-9188 | Fax: 1-732-210-0262 | Email: info@genscript.com 5
You can also read
