Standardisation

Question 1

Define standardisation

Answer 1

The process of developing and implementing technical standards

Question 2

What is a standard?

Answer 2

An agreed way of doing something eg. making a product, measuring something, delivering a service

Question 3

Why is standardisation necessary?

Answer 3

To provide a reliable basis for people to share the same expectations about a product or service

Question 4

What are the advantages of standardisation?

Answer 4

To facilitate trade, to provide a framework for achieving economies, efficiencies and interoperability, to enhance consumer protection and confidence

Question 5

What is the advantage of a standardised visual description?

Answer 5

You do not need to have a common language to understand it.

Question 6

What are the two types of standardisation in synthetic biology?

Answer 6

Standardisation of functional composition

Standardisation of physical composition

Question 7

Define functional composition standardisation

Answer 7

To group parts of similar functions together

Question 8

Name the 5 advantages of functional composition

Answer 8

1. Ignore the complicated names of parts
2. Document a list (catalogue) of parts into parts registries
3. Links with the characteristics of the parts
4. Allow the designer to mix and match parts
5. Allows communication of design in an accessible way

Question 9

What does SBOL stand for?

Answer 9

Synthetic biology open language

Question 10

What does the promotor symbol look like?

Answer 10

A 90-degree bent thin arrow sitting on the line

Question 11

What does the ribosome entry site symbol look like?

Answer 11

A semi-circle sitting on the line

Question 12

What does the cds (codon sequence) symbol look like?

Answer 12

A thick right-directional arrow replacing the line

Question 13

What does the terminator symbol look like?

Answer 13

A large T sitting on the line

Question 14

What does an operator symbol look like?

Answer 14

A box replacing the line.

Question 15

What does an assembly scar symbol look like?

Answer 15

Two horizontal lines of equal length

Question 16

What does the mRNA symbol look like?

Answer 16

One squiggly line

Question 17

Define physical composition standardisation

Answer 17

to describe how the parts can be linked to form devices systems - pre-determined and predictable (assembly standards)

Question 18

What are the advantages of physical composition standardisation?

Answer 18

1. Compatibility of parts

2. Accommodate increasing complexity in design

Question 19

Name the key characteristic of assembly standard

Answer 19

Indempotence

Question 20

Define indempotency

Answer 20

An operation that produces the same results no matter how many times it is performed

Question 21

Explain how indempotency works in synthetic biology.

Answer 21

In rounds of increasingly complex assembly, the assembled parts always adhere to the assembly requirements

Question 22

Describe indempotence in plumbing

Answer 22

No matter how many pipes you put together using the parts, the ends always have screw threads allowing sequential assembly.

Question 23

Describe traditional cloning

Answer 23

A piece of donor DNA is cleaved (cut) using a restriction enzyme. There is not usually a clean break so there will be some overhang from one strand on either end.
A vector plasmid (a small, single stranded circular piece of DNA not found in the chromosome) are also cut using the same restriction enzyme.
The end of the vector plasmid and the overhand from the donor DNA anneal with the help of an enzyme called DNA ligase (ligation).
This is then inserted into the organism of choice.

Question 24

What are the disadvantages of traditional cloning?

Answer 24

There is not enough detail provided, you are free to choose whichever restriction sites and plasmid DNA you want.
The methodology will be different from lab to lab so the work is very difficult to replicate.
There is no indempotence.
A very in-depth microbiology knowledge is needed.

Question 25

What is the more common name for the most used standardised physical composition technique?

Answer 25

The BioBrick technique or RFC[10]

Question 26

What are BioBrick Prefix and Suffix

Answer 26

The prefix and suffix are each two sequences of DNA to which the restriction enzyme can attach and break the DNA into two.

Question 27

When is a part called a BioBrick?

Answer 27

Only when the prefix and the suffix are attached to the part

Question 28

What does a plasmid used for BioBricks contain apart from the prefix and suffix?

Answer 28

The ‘origin’ of replication that allowws the plasmid to replicate within the host of choice.
It also has a resistance cassette, which is resistant to a particular antibiotic.

Question 29

Why do you need a resistance cassette in the BioBrick plasmid backbone?

Answer 29

It allows you to identify between the organisms that have that particular plasmid to the ones that don’t, by exposing them to the relevant antibiotic. Only the ones that have the desired plasmid will survive.

Question 30

Describe how to assemble two parts together using the RFC[10] process

Answer 30

Choose two BioBricks from the parts registry and cut in the desired places using the corresponsing restriction enzymes. The part you want to insert cut at the E prefix and S suffix locations. The BioBrick into which you desire to insert the part is cut at the E and X locations. The overhang of the DNA on the S end of the part and the overhang of the X end of the BioBrick are complementary and will aneal toether in the presence of a ligase enzyme. This creates a mixed scar site (M) that is not recognised by Xba1 or Spe1 as it contains the stop codon TAG that causes the ribosome to detach from the mRNA.

Question 31

Name 3 advantages of the RFC[10] process

Answer 31

Standard, well tested and documented, large and still growing set of parts

Question 32

Name 2 disadvantages of the RFC[10] process

Answer 32

Cannot be used for protein fusion due to the stop codon between parts
Cannot be used if the parts contain the same restriction sites that are in the prefix or the suffix

Question 33

Give an example of where you might want protein fusion.

Answer 33

If you want an antibody to recognise both a healthy and a diseased state.

Question 34

Give a naturally occuring example of protein fusion.

Answer 34

Leukemia is a fusion of proteins from chromosome 22 and chromosome 9

Question 35

Which is the most used assembly method in synthetic biology?

Answer 35

Gibson Assembly

Question 36

What is the assembly format of RCF[10]?

Answer 36

Restriction-ligation

Question 37

What is the assembly format of Gibson assembly?

Answer 37

Polymerase-nuclease-ligase

Question 38

Describe the Gibson assembly method.

Answer 38

Begin with two parts or DNA fragments that you wish to join together. You add an overlap by adding PCR
The stitched DNA fragments can then be retrieved from the vial.

Question 39

Describe the Gibson assembly method.

Answer 39

Begin with two parts or DNA fragments that you wish to join together. You add a 15-20 bp overlap by adding PCR.
Add these overlapped DNA to a vial containing the Gibson Assembly Master Mix (your vector plasmid and 3 enzymes). Incubate this at 50C.
Exonuclease will sequentially cleave nucleotides from one end of the 5’ strand of both of your DNA fragments. The fragments will then anneal.
DNA polymerase will then close the gaps by adding nucleotides back onto the cleaved ends, and DNA ligase will seal the nicks.
The stitched DNA fragments can then be retrieved from the vial.

Question 40

What are the three enzymes you need for the Gibson assembly method?

Answer 40

Exonuclease, DNA polymerase and DNA ligase

Question 41

How long does the Gibson assembly method take?

Answer 41

Around an hour