Plasmids 101: What is a Plasmid?

Question 1

What are plasmids at the most basic level?

Answer 1

At their most basic level, plasmids are small circular pieces of DNA that replicate independently from the host’s chromosomal DNA.

Question 2

Where are plasmids mainly found?

Answer 2

They are mainly found in bacteria, but also exist naturally in archaea and eukaryotes such as yeast and plants

Question 3

What do all natural plasmids contain?

Answer 3

All natural plasmids contain an origin of replication (which controls the host range and copy number of the plasmid) and typically include a gene that is advantageous for survival, such as an antibiotic resistance gene.

Question 4

How do plasmids utilised in the lab differ to those found in nature?

Answer 4

In contrast, plasmids utilized in the lab are usually artificial and designed to introduce foreign DNA into another cell. Minimally, lab-created plasmids have an origin of replication, selection marker, and cloning site. The ease of modifying plasmids and the ability of plasmids to self-replicate within a cell make them attractive tools for the life scientist or bioengineer.

Question 5

Describe the origin of replication vector element

Answer 5

DNA sequence which allows initiation of replication within a plasmid by recruiting replication machinery proteins

Question 6

Describe the Antibiotic Resistance Gene vector element

Answer 6

Allows for selection of plasmid-containing bacteria.

Question 7

Describe the Multiple Cloning Site (MCS) vector element

Answer 7

Short segment of DNA which contains several restriction sites allowing for the easy insertion of DNA. In expression plasmids, the MCS is often downstream from a promoter.

Question 8

Describe the Insert vector element

Answer 8

Gene, promoter or other DNA fragment cloned into the MCS for further study.

Question 9

Describe the Promoter Region vector element

Answer 9

Drives transcription of the target gene. Vital component for expression vectors: determines which cell types the gene is expressed in and amount of recombinant protein obtained.

Question 10

Describe the Selectable Marker vector element

Answer 10

The antibiotic resistance gene allows for selection in bacteria. However, many plasmids also have selectable markers for use in other cell types.

Question 11

Describe the Primer Binding Site vector element

Answer 11

A short single-stranded DNA sequence used as an initiation point for PCR amplification or sequencing. Primers can be exploited for sequence verification of plasmids.

Question 12

How are artificial plasmids often referred to?

Answer 12

Due to their artificial nature, lab plasmids are commonly referred to as “vectors” or “constructs.”

Question 13

How may a gene of interest be inserted into a vector?

Answer 13

To insert a gene of interest into a vector, scientists may utilize one of a variety of cloning methods (restriction enzyme, ligation independent, Gateway, Gibson, etc). The cloning method is ultimately chosen based on the plasmid you want to clone into. Regardless, once the cloning steps are complete, the vector containing the newly inserted gene is transformed into bacterial cells and selectively grown on antibiotic plates. Importantly, because the bacteria from which plasmids are isolated grow quickly and make more of the plasmids as they grow, scientists can easily make large amounts of plasmid to manipulate and use in later work.

Question 14

How do scientists use plasmids?

Answer 14

Generally, scientists use plasmids to manipulate gene expression in target cells. Characteristics such as flexibility, versatility, safety, and cost-effectiveness enable molecular biologists to broadly utilize plasmids across a wide range of applications

Question 15

Give six types of common plasmid

Answer 15

Some common plasmid types include
- cloning plasmids
- expression plasmids
- gene knock-down plasmids
- reporter plasmids
- viral plasmids
- genome engineering plasmids.

Question 16

Name five things plasmids can be used for

Answer 16

Produce large amounts of a protein so that scientists can purify and study it in a controlled setting.

Produce proteins that glow so that scientists can track their location or quantity inside a cell

Monitor the level of a chemical in a particular environment

Produce enzymes that will make specific, controlled changes to an organism’s genome (genome engineering)

Produce synthetic viruses that can be used in research or for therapeutics

Question 17

What is another term for the origen of replication?

Answer 17

Replicon

Question 18

What is the replicon comprised of?

Answer 18

The replicon is comprised of the origin of replication (ORI) and all of its control elements.

Question 19

What happens at the ORI?

Answer 19

The ORI is the place where DNA replication begins, enabling a plasmid to reproduce itself as it must to survive within cells

Question 20

How do replicons of plasmids compare to those used to replicate chromosomal DNA?

Answer 20

The replicons of plasmids are generally different from those used to replicate the host’s chromosomal DNA, but they still rely on the host machinery to make additional copies.

Question 21

Describe ORI sequences in terms of their nucleotides and the functional relevance

Answer 21

ORI sequences are generally high in As and Ts; A-T base pairs are held together with two hydrogen bonds instead of three as G-C pairs are. As a result, stretches of DNA that are rich in A-T pairs melt
more readily at lower temperatures. When DNA melts, it gives the replication machinery room to come in and get busy making copies.

Question 22

How are all ORIs not created equal?

Answer 22

Some will produce many plasmid copies and others produce just a few copies depending on how they are regulated.

Question 23

What is meant by the terms ‘relaxed’ and ‘stringent’ in terms of ORIs?

Answer 23

Generally, control of replication is referred to as “relaxed” or “stringent” depending on whether the ORI is positively regulated by RNA or proteins, respectively.

Question 24

What intracellular factors influence a plasmids copy number?

Answer 24

A plasmid’s copy number has to do with the balance between positive and
negative regulation and can be manipulated with mutations in the replicon. For example, the pMB1
ORI maintains about 20 copies per cell, while pUC – which differs by only two mutations – will produce as many as 700 copies per cell.

Question 25

What three questions should you ask yourself when deciding on an ORI?

Answer 25

• Will the plasmid be used exclusively in E. coli? Gram negative bacteria in general? Both gram negatives and gram positives?
• Will you have only one plasmid type in your cells at a time?
• Do you want to make a lot of your plasmid? Is the gene toxic in high amounts?

Question 26

What is it good to keep in mind regarding plasmids with low copy numbers?

Answer 26

It is always good to keep in mind that plasmids with low to medium copy numbers can still express massive amounts of protein given the proper promoter (stay tuned!) and growth conditions

Question 27

How is compatibility related to the similarity of ORIs in vectors?

Answer 27

Generally speaking, plasmids with the same ORIs are incompatible because they will compete for the same machinery, creating an unstable and unpredictable environment. As a rule, plasmids from the same group should not be co-transformed, so if you require two plasmids for an experiment, make sure they have “compatible” ORIs

Question 28

Where can you find whether common vectors are in the same compatibility groups?

Answer 28

Table 1.2 highlights common cloning vectors, their copy number, ORI, and incompatibility group.

Question 29

What other factors influence copy number? (4)

Answer 29

The insert
Bacteria tend to maintain fewer copies of plasmids if they contain large inserts or genes that create a toxic product.

The E. coli strain
Most E. coli strains can be used to propagate plasmids, but end A- E. coli are best for high yields of plasmids.

Growth conditions
The amount of aeration, temperature, culture volume, antibiotic, and medium can all affect copy number. Some ORIs are temperature sensitive.

The Culture Inoculum
Freshly streaked bacteria have higher copy numbers – for optimal results always pick a single colony and do not subculture directly from glycerol
stocks, agar stabs, or liquid cultures.

Question 30

Regarding growth conditions, how can you ‘trick’ ORIs into amplifying more copies?

Answer 30

Some ORIs are temperature sensitive; other ORIs can be “tricked” into amplifying more copies with the addition of Chloramphenicol – make sure your growth conditions aren’t working against you!

Question 31

What length of incubation time tends to give higher copy numbers and why?

Answer 31

Incubation for 12-16 hours tends to give higher copy numbers since the bacteria have just reached stationary phase, but the cells have not started to die.

Question 32

In practice, what does the term ‘promoter’ describe?

Answer 32

In practice, the term “promoter” describes the combination of the promoter (RNA polymerase binding site) and operators (response elements).

Question 33

How long typically are promoters? Where are they located?

Answer 33

Promoters are about 100 to 1000 base pairs long and found upstream of their target genes.

Question 34

What does the sequence of the promoter region control?

Answer 34

The sequence of the promoter region controls the binding of the RNA polymerase and transcription factors, therefore promoters play a large role in determining where and when your gene of interest will be expressed.

Question 35

How is RNA transcribed from DNA?

Answer 35

RNA is transcribed from DNA using an RNA polymerase (RNAP).

Question 36

How many enzymes are responsible for this transcription in bacteria?

Answer 36

In bacteria, this is done by a single enzyme; however, eukaryotes have multiple polymerases which are each responsible for a specific subset of RNAs.

Question 37

What relevance does the specificity of RNAP have for your promoter in terms of RNA? Give two broad examples

Answer 37

To gain this specificity, the eukaryotic RNAP can recognize and bind to specific promoter elements. This means that the promoter present in your plasmid backbone must be compatible with the type of RNA that needs to be made: if you want mRNA (for gene expression) you need to use an RNAP II promoter, whereas small RNAs (such as shRNA) are transcribed from the RNAP III promoters.

Question 38

What else in there to be considered in regards to promotor specificity?

Answer 38

You will also need to make sure your plasmid has a promoter suited to working in your host organism. Because transcription machinery differs between cell types or organisms, promoters must be similarly variable. Bacterial promoters only work in prokaryotic cells and typically only in the same or closely related species from which they were derived. Similarly, the various eukaryotic cell types (mammalian, yeast, plants, etc.) require unique promoters and there is very little crossover.

Question 39

Compare bacterial and eukaryotic promotors in a general sense

Answer 39

Generally speaking, promoters in bacteria are less diverse and complex, having fewer parts than those in eukaryotic cells. Some promoters are constitutively active and on all the time, while others are more carefully controlled.

Question 40

How may regulated promotors be regulated?

Answer 40

Regulated promoters might act only in certain tissues or at certain times in development or there may be ways to turn them on or off at will with
a chemical, heat, or light. In the cell, promoters themselves are controlled by still other regulatory factors: enhancers, boundary elements, insulators, and silencers

Question 41

What may occur in these regulated promotors which might not cause issues in a cell but can confound research results?

Answer 41

Some “leaky” transcription may occur. This is normally not a big issue
for cells, but it may confound research results or even kill your cells if your gene of interest is toxic.

Question 42

How have scientists found a way to combat leaky transcription?

Answer 42

To combat this, scientists have created synthetic promoters, which typically include some combination of other promoter elements, and tend to be more tightly regulated.

Question 43

Where can you get some insight into commonly used promotors?

Answer 43

Tables 1.3 and 1.4 list some of the most common bacterial and mammalian promoters.

Question 44

Plasmids designed to express genes in a given host cell type are generally broken down into which two broad categories based on what characteristics?

Answer 44

Prokaryotic or eukaryotic, based on the functional elements they contain. Plasmid DNA in both prokaryotic and eukaryotic systems must be transcribed into RNA.

Question 45

In what stages does transcription occur?

Answer 45

Transcription occurs in three phases: initiation, elongation, and termination.

Question 46

What element is involved in the initiation steps of gene transcription?

Answer 46

The promoter

Question 47

What element is involved in terminating gene transcription and what is involved in this

Answer 47

The role of the terminator, which is a sequence-based element, is to define the end of a transcriptional unit (such as a gene) and initiate the process of releasing the newly synthesized RNA from the transcription machinery.

Question 48

Describe the typical location of terminators

Answer 48

Terminators are found downstream of the gene to be transcribed, and typically occur directly after any 3’ regulatory elements, such as the polyadenylation or poly(A) signal.

Question 49

What role is greatly studied in gene promoters and often neglected in terminators?

Answer 49

While many studies focus on promoter strength as a determinant of gene expression levels, the terminator also plays an important role in RNA processing and contributes to variability in RNA half-life, and ultimately gene expression.

Question 50

What is meant by polyadenylation?

Answer 50

Polyadenylation, as the name implies, is the post-transcriptional additional of multiple adenine (A) nucleotides to the tail of a messenger RNA transcript.

Question 51

What is the purpose and mechanism of polyadenylation?

Answer 51

The purpose and mechanism of polyadenylation vary among cell types, but polyadenylation generally serves to promote transcript longevity in eukaryotes and promote transcript degradation in prokaryotes.

Question 52

What two categories do prokaryotic termination mechanisms typically fall under?

Answer 52

rho-dependent and rhoindependent

Question 53

What is meant by this rho dependence? Which are more common in bacterial expression plasmids?

Answer 53

Rho factor is a helicase which assists RNA polymerase in the termination of the
transcript. Rho-dependent terminators are not usually employed in plasmid-based expression systems, so these will not be detailed here. Nearly all common bacterial expression plasmids
use Rho-independent terminators.

Question 54

Are rho-independent terminators naturally occuring or engineered? Give examples

Answer 54

They include naturally occurring terminators, such as T7 and rrnB, as well as engineered high-efficiency terminators such as T0.

Question 55

What is rho-independent termination also known as and what does it rely on?

Answer 55

Rho-independent termination
is also known as intrinsic termination, and relies on the formation of a GC-rich hairpin in the RNA transcript followed by a weakly bound poly-uracil tract.

Question 56

What is the purpose of the tertiary structure of the intrinsic termination?

Answer 56

The tertiary structure of the hairpin-DNA complex is thought to destabilise the transcription complex, initiating cleavage of the transcript.

Question 57

How efficient are terminators at halting transcription?

Answer 57

No terminator is 100% efficient at halting
transcription of the template and initiating the desired cleavage event, although some engineered terminators come close (>95%). For most purposes, however, any common terminator will suffice.

Question 58

How do many commercial vectors reduce unwanted translation of downstream elements?

Answer 58

Many commercial expression vectors use double terminators to reduce unwanted translation of downstream elements.

Question 59

When might a high affinity terminator be desired?

Answer 59

A high affinity terminator may be desired for multicistronic constructs where high termination efficiency is necessary to minimize transcriptional read-through. Multicistronic or bicistronic vectors contain IRES and/or 2A peptides to allow for coexpression of multiple genes from a single transcript.

Question 60

How does the poly(A) tails of prolaryotic transcripts differ to that of eukaryotic transcripts?

Answer 60

Although mostly thought of as a eukaryoticspecific process, prokaryotes also add poly(A) tails to certain RNAs. Unlike the eukaryotic mechanism
which requires a consensus sequence for the addition of a poly(A) tail, the addition of a poly(A) tail on a prokaryotic transcript is non-specific and can be added to any accessible 3’ end.

The presence of the poly(A) tail targets the RNA to the degradosome, which contains enzymes that cut RNA not protected by secondary structure.

Question 61

What is meant by a consensus sequence?

Answer 61

In molecular biology and bioinformatics, the consensus sequence is the calculated sequence of most frequent residues, either nucleotide or amino acid, found at each position in a sequence alignment. It represents the results of multiple sequence alignments in which related sequences are compared to each other and similar sequence motifs are calculated.

Question 62

What is thought about the function of poly(A)s in prokaryotes due to their lack of specificity?

Answer 62

Because it lacks specificity, it is thought that poly(A)s are used to control the cellular concentration of regulatory RNAs and may additionally act as a quality control mechanism to rid the cell of misfolded RNAs.

Question 63

How many RNA polymerases do eukaryotes have?

Answer 63

Unlike prokaryotes that have a single RNA polymerase for transcription, eukaryotes have three RNA polymerases (Polymerases I, II, and III)

Question 64

What are the roles of the different polymerases?

Answer 64

Each are responsible for transcribing different types of RNA: Polymerase I is responsible for ribosomal RNA, Polymerase II is responsible for mRNA and miRNAs, and Polymerase III transcribes tRNA and other short RNAs.

Question 65

Do the different polymerases differ in their termination?

Answer 65

Although not as well studied as prokaryotic termination, the basic processes for eukaryotic termination are understood and it has been noted that each eukaryotic RNA polymerase terminates differently.

Question 66

Describe one aspect in which Polymerase III differs to the other two in termination

Answer 66

Polymerase III, for example, relies on a specific sequence and RNA secondary structure to induce transcript cleavage, similar to the Rho-independent termination found in prokaryotes. This is different than Polymerases I and II, which both require binding of termination factors.

Question 67

How is the termination of polymerase I and II different from one another?

Answer 67

Although both are termination factor dependent, Polymerases I and II employ different mechanisms to terminate transcription. Polymerase I uses a
process similar to the prokaryotic Rho-dependent mechanism, whereas Polymerase II termination is more complex and involves two RNA polymerase-associated proteins, CPSF and CstF, which are responsible for recruiting the cleavage and
polyadenylation enzymes, in a process that seems to couple termination with polyadenlyation.

Question 68

What are mammalian expression plasmids primarily used for and and what are the commonly used mammalian terminators?

Answer 68

Mammalian expression plasmids are primarily used to create mRNA and the commonly used mammalian terminators (SV40, hGH, BGH, and rbGlob) include the sequence motif AAUAAA which promotes both polyadenylation and termination.

Question 69

Out of those listed, which of the mammalian terminators are thought to be most efficient in stopping transcription?

Answer 69

Out of those listed, the SV40 late polyA and rbGlob polyA are thought to be more efficient in terminating transcription due to the presence of additional helper sequences

Question 70

As alluded to above, termination and polyadenylation of Polymerase II transcripts (and therefore mRNAs) are coordinated processes. Briefly describe this

Answer 70

Cleavage between the consensus motif and a downstream GU-rich region (shown in the figure below) releases the mRNA from the polymerase and creates a
free 3’ end which is now available for polyadenylation. The addition of the poly(A) tail is important for stability of the mRNA, protection from degredation, and is integral to the nuclear export and translation processes as well.

Question 71

The textbook poses the following question:

“Have you ever tried digesting with XbaI or ClaI restriction enzymes and gotten unusual or unexpected results? Or considered why DpnI will degrade your
template DNA from a PCR reaction but not the newly synthesized product from a site-directed mutagenesis experiment?”

What does the book pose as the answer to both of these questions?

Answer 71

methylation

Question 71

Why does methylation occur?

Answer 71

Aside from restriction modification systems, DNA methylation also plays an integral role in regulating genome replication, repairing mismatched basepairs or small indels that occur during DNA synthesis, and promoting or repressing protein expression.

Question 72

What are restriction modification systems?

Answer 72

The restriction modification system is found in bacteria and archaea, and provides a defense against foreign DNA, such as that borne by bacteriophages. Bacteria have restriction enzymes, also called restriction endonucleases, which cleave double-stranded DNA at specific points into fragments, which are then degraded further by other endonucleases.