pBluescript is an example of a combination between plasmids and phages (phagemids). Phagemids represent a hybrid type of class of vectors that serve to produce single-stranded DNA.

1. pBLUESCRIPT

2. pBluescript is an example of a combination between
plasmids and phages (phagemids).
Phagemids represent a hybrid type of class of vectors that
serve to produce single-stranded DNA.
Features
1. Like pUC vectors, which pBluescript is derived from,
there is a multiple cloning site inserted into the LacZ'
gene
2. pBluescript also contains the origin of replication of the
single-stranded phage f1, which is related to M13 phage
vectors. This translates into that a cell harboring a
recombinant phagemid, if infected by f1 helper phage
that supplies the single-stranded phage DNA replication
components, it will produce and package single-stranded
phagemid DNA.

4. 3. Produce RNA transcripts
pBS has a T3 promoter on one end side and a T7 promoter
on the other terminal end.
This is key because it enables one to isolate the double-
stranded phagemid DNA and transcribe it in vitro with either
of these two phage polymerases to produce pure RNA
transcripts to coincide with either of the two different
strands

5. • 2961 basepair plasmid
• Derived By Replacing pUC19 polylinker of pBS (+/-) with
synthetic polylinker
• SK designation indicates that the polylinker is oriented such
that the lacZ transcription proceeds from Sac I to Kpn I.
• 21 unique restriction sites in the multiple cloning region
• Blue/White color selection
• In vitro RNA transcription with T3 or T7 RNA polymerase
•Double and Single-stranded sequencing
Summary-pBluescript

6. pBluescript II SK, either the + or - orientation.
The + and - denote which single strand of DNA, either the
sense or anti-sense strand, can be rescued using a helper
phage.
These + or - orientations occur in the f1 region of the plasmid.
Thus:
f1 (+) origin: (consists of 3-459 base pairs) f1 filamentous
phage origin of replication that allows for the recovery of the
SENSE strand of the lacZ gene
f1 (-) origin: (consists of 3-459 base pairs) f1 filamentous
phage origin of replication allowing recovery of the ANTISENSE
strand

7. • Extensive set of restriction sites in polylinker, many with unique 4-base
overhangs
• Blue/white section
• Opposing T7promoters for making RNA transcripts in either direction or
double-stranded RNA
• Single-stranded (M13) DNA replication
origin.
• Compatible with pUC/M13
sequencing primers
pLITMUS 28i and 38i

8. GATEWAY Cloning Technology
The GATEWAY Cloning Technology is based on the site-specific
recombination system used by phage l to integrate its DNA in the E. coli
chromosome. Both organisms have specific recombination sites called attP
in phage l site and attB in E. coli.
The integration process (lysogeny) is catalyzed by 2 enzymes: the phage l
encoded protein Int (Integrase) and the E. coli protein IHF (Integration
Host Factor).
Upon integration, the recombination between attB (25 nt) and attP (243 nt)
sites generate attL (100 nt) and attR (168 nt) sites that flank the integrated
phage l DNA
The process is reversible and the excision is again catalyzed Int and IHF in
combination with the phage l protein Xis.
The attL and attR sites surrounding the inserted phage DNA recombine
site-specifically during the excision event to reform the attP site in phage l
and the attB site in the E. coli chromosome.

10. The GATEWAY reactions are in vitro versions of the
integration and excision reactions.
To make the reactions directional two slightly different and
specific site were developed, att1 and att2 for each
recombination site.
These sites react very specifically with each other.
For instance in the BP Reaction attB1 only reacts with attP1
resulting in attL1 and attR1, and attB2 only with attP2 giving
attL2 and attR2.
The reverse reaction (LR Reaction) shows the same
specificity.

12. Step 1 Cloning the gene of interest into an Entry Vector
using the BP Reaction.
Step 2 Subcloning the gene of interest from the Entry Clone
(Step 1) into a Destination Vector using the LR
Reaction producing the Expression Clone.
The ultimate goal of the GATEWAY reactions is to make
an expression clone.
This is often a two step process:

13. The gene of interest is cloned into an Entry Vector and flanked
by the attL1 and attL2 recombination sites.
The Entry Vector is transcriptionally silent and contains the
gene for kanamycin resistance (Kmr).
To produce the Expression Clone the gene has to be subcloned
into a Destination Vector that contains all the sequence
information necessary for expression
• the gene for ampicillin resistance (Apr),
• and two recombination sites (attR1 and attR2) that flank a
• gene for negative selection ccdB (the encoded protein is toxic
for the standard E. coli strains).

14. The two plasmids are mixed and the LR CLONASE Enzyme
Mix is added.
The reaction is directional and specific, so that attL1 only
reacts with attR1 and attL2 with attR2.
The recombination yields two constructs: the intended
Expression Clone and a by-product (Donor Vector).
The produced expression clone is under two forms of
selection: the antibiotic resistance and the negative selection
by the toxic ccdB protein.
High levels of positive clones (typicaly more than 99%) are
obtained after transformation to a standard cloning or
expression strain like DH5a or BL21

15. One of the main advantages of the GATEWAY Cloning Technology is that once you
have made an Entry Clone the gene of interest can be easily subcloned into a wide
variety of Destination Vectors using the LR Reaction

16. TA Cloning exploits the terminal transferase activity of some DNA
polymerases such as Taq polymerase.
This enzyme adds a single, 3'-A overhang to each end of the PCR
product.
This makes it possible to clone this PCR product directly into a
linearized cloning vector with single, 3'-T overhangs.
This polymerase lacks 3' to 5'
proofreading activity adds a single,
3'-adenine overhang to each end of
the PCR product.
It is best if the PCR primers have
guanines at the 5' end as this
maximizes probability of adding the
terminal adenosine overhang
TA Cloning Vectors

17. The pGEM -T Easy Vector multiple cloning region is flanked by
recognition sites for the restriction enzymes EcoRI, BstZI and NotI, providing
three single-enzyme digestions for release of the insert.
pGEM -T and pGEM -T Easy

18. The only difference between pGEM-T and pGEM-T Easy is in the
multiple cloning site (MCS).
The MCS of the pGEM-T Easy Vector contains sequences on
either side of the insert that are recognized by the restriction
enzymes Not I and EcoR I.
This allows the insert DNA to be removed with a single
restriction digest using either of these enzymes.

20. pCAMBIA

21.  high copy number in E.coli for high DNA yields
 pVS1 replicon (replicon of the 29kbp of Pseudomonas plasmid pVS1)
for high stability in Agrobacterium
 small size, 7-12kb depending on which plasmid
 restriction sites designed for modular plasmid modifications and
small but adequate poly-linkers for introducing your DNA of
interest
 bacterial selection with chloramphenicol or kanamycin
 plant selection with hygromycin B or kanamycin
 simple means to construct translational fusions to gusA reporter
genes.

22. GUS reporter system
The activity of a promoter (in terms of expression of
a gene under that promoter) either in a quantitative
way or through visualization
The technique is based on β-glucuronidase, an
enzyme from the bacterium Escherichia coli
When incubated with some specific colorless or non-
fluorescent substrates, can transform them into
colored or fluorescent products

23. There are different possible glucuronides that can be used as
substrates, depending on the type of detection needed
(histochemical, spectrophotometrical, fluorimetrical).
The most common substrate for GUS histochemical staining is
5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc): the product
of the reaction is in this case a clear blue color.
Other common substrates are p-nitrophenyl β-D-glucuronide
for the spectrophotometrical assay and 4-methylumbelliferyl-
beta-D-glucuronide (MUG) for the fluorimetrical assay