Dr.S.

Geetha Priya

Unit 4: Modern Biotechnological Processes

Guidelines for choosing Host-Vector Systems

 Selecting the right vector for your molecular biology experiment is

fundamental to ensure the experiments success. There are many elements to
consider when choosing which vector to use.

 The first question to ask yourself is “What you intend to do with your vector ?”.

Types of Vectors

Vectors are broadly classified into two types namely (a) Cloning Vectors and (b)
Expression Vectors.

a) Cloning Vectors are useful for generating many copies of the gene. It usually
contains features associated with the insertion or removal of DNA fragments.
For example, it has multiple cloning sites with many restriction sites, antibiotic
resistant genes, etc. Cloning vectors provide a basic backbone for the DNA
insert to be reproduced and generally have the common features just
described, but these vectors are useful only for cloning and not for expressing
a protein product. The DNA insert in a cloning vector can be copied but not
translated into a functional protein product.

b) Expression Vectors are associated with the actual expression of the gene
into mRNA and protein in the target organism. Expression vectors contain
additional organism specific sequences relating to expression such as
promoters, RBS sequences, Kozak sequences (in eukaryotes), or the Shine
Dalgarno sequence (in prokaryotes). For a gene to give rise to its protein
product, an expression vector must be used that contains the appropriate
pieces for a host cell to produce the protein. In the case of a mammalian cell,
a standard mammalian expression vector will still contain the origin of
replication, multiple cloning sites, and selectable marker, but it will also need a
promoter found in mammalian cells that can drive the expression of the gene,
as well as other standard features found in genes. Expression vectors need to
be transcribed and translated, so they need the polyadenylation tail that
normally appears at the end of transcribed pre-mRNA to protect and stabilize
it, a minimal untranslated region (UTR) so that there is not an excess of DNA
that will not be translated, and a sequence that attracts the ribosome for
translation.

Vector Size

 Vector Size is relevant for both Cloning and Expression Vectors. The only
aspect to consider here is whether you are cloning a larger or smaller DNA
fragment.

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 Most general plasmids (i.e. pUC) can cope with inserts upto 15 kb. Anything
bigger can complicate the replication and cause problems with stability.
However, if you have a large DNA insert, there are special types of vectors
suitable for such applications, which have a different “Origin of replication”. As
a general remark, “Origin of replication” of bigger vectors have a lower copy
number than those of smaller vectors.

 Bigger the vector, lower is the transfection or transformation efficiency and its
stability. Nevertheless, vectors can usually go upto 52 kb.

Selectable Markers

 Both Cloning and Expression Vectors require you to choose a selectable

marker. This marker allows for the identification of a positive transformant.
When you grow your cell lines under this condition, only the cells that have
incorporated the vector will survive (a process called selection).

 There are two major types of selectable markers

1. Drug-resistance markers: incorporation of a gene encoding an enzyme that

inactivates a specific antibiotic.

2. Auxotrophic markers: allows cells with the marker to survive without

essential nutrients in the medium.

3. When choosing your cloning vector resistance, you should consider using:

a) A positive selection - under selection (antibiotic or nutrient), only cells

that incorporated the selectable marker will survive.

b) A negative selection - under selection, only cells that did not

incorporate the selectable marker will survive.

Multiple Cloning Sites (MCS)

 A crucial step during the cloning process is the ligation of the DNA fragment to
the plasmid, which is greatly facilitated by the use of specific restriction
enzymes. This means that it is important to check if the desired restriction
sites are compatible with your insert.

 Most modern vectors include an artificial stretch of DNA called MCS

containing many different long restriction endonuclease cutting sites.

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Preventing Self-ligation

 The presence of sticky ends in vectors after the digest process often causes
self-ligation. In order to avoid this issue, you can use a phosphatase treatment
just before the ligation step i.e., dephosphorylate the vector using alkaline
phosphatase. In this process the 5’ phosphate group, which the ligase needs
for phosphodiester bond formation, is removed.

For Cloning Vectors Only

Copy Number

 If you choose to work with a cloning vector, you need to decide what is the
copy number (high, medium, or low) in order to receive the desired number of
copies at the end of the process.

 Usually, a high-copy vector is the best approach to produce the highest yields.
For example, pUC has a copy number of 700.

For Expression Vectors Only

Organism

 If you want to drive the expression of your desired gene, you need a vector
that contains functional elements in your host organism.

 Expression vectors produce proteins through transcription of the vector's

insert followed by translation of the mRNA produced.

 Although they share similar requirements, expression in different host

organisms require additional elements: a selectable marker, a promoter for
initiation of transcription, a ribosomal binding site for translation initiation, a
termination signal, and so on.

 The common organisms used for high protein expression are:

1. Mammalian cells usually CHO (Chinese Hamster Ovary) cells
2. Insect cells using the Baculovirus expression system
3. Pichia pastoris, single cell eukaryotic fungi with glycosylation, specially
suited for large scale protein production
4. Saccharomyces cerevisiae, the workhorse of every eukaryotic research
5. Escherichia coli, the workhorse of prokaryotic research
6. Plants cells usually “Tobacco Cell Culture”
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