Genome techniques Flashcards
(48 cards)
What is a plasmid?
A plasmid is a small circular piece of DNA that grows on bacteria independent from the bacterial chromosome.
What is the basic cloning technique?
We want to clone a specific part of a DNA sequence by transferring it to a cloning vector. We add the fragment to a DNA vector to get a recombinant vector. The vector is transferred to a bacterial plasmid and into a host cell where it will now transcribe the DNA we want.
What is the BioBrick hierarchy?
System, device, part
How does BioBrick work?
We want to combine two BioBrick parts so two pieces of DNA inside a plasmid each. The brick is flanked by two restriction sites which when cleaved gives an overhang of sticky ends. Depending on which brick should go into which brick we can cleave them differently, but they will create a ‘scar’ that can no longer be recognized by the restriction enzymes (aka uncleavable). We end up with a single BioBrick that is still flanked with the restriction sites but not in between the parts. The process can be iterated.
What is a scar in cloning/assembly and why can it be bad
A scar is specifically used in BioBricks to be between the bricks to ensure no cleavage from the restriction sites. It can be bad if the scar becomes a coding part.
What is a restriction enzyme?
A restriction enzyme is a protein isolated from bacteria that cleaves DNA sequences at sequence-specific sites, producing DNA fragments with a known sequence at each end
What is a fusion site?
Unique overhangs (often 4 bases)
What is a palindromic sequence?
Reverse complement strands
What is an amplicon?
Source/product of amplification/replication. Often PCR.
What is the advantage of using Golden Gate shuffling instead of BioBrick?
In Golden Gate we can introduce several fragments at a time without undergoing iterative manual processes.
What is the purpose of Golden Gate shuffling?
To assemble several fragments in a single cloning reaction.
What is the process of Golden Gate shuffling?
We have a destination vector and one or more fragments. On the destination vector we have a recognition site (non-palindromic 4-7 nucleotides). We use type IIS enzymes that gives a 4 base nucleotide overhang called fusion sites.
Its important that the fusion sites are facing away from each other in the destination vector because when we then remove the target, the plasmid will not recirculate.
The target vector:
We can use any type of fragment for the assembly including plasmids and PCR products. We can design PCR primers with flanking bases, type IIS recognition sites and an overhang sequence. This way we can introduce the recognition sites on each side of the fragment (flanking the fragment). This is called tail-PCR.
Our fusion sites should be facing inwards so cut the two recognition sites and get the target sequence out.
Once both target fragment and destination vector is ready they are mixed in a reaction with type IIS enzymes and DNA ligase. We are cleaving inside the fragment so the recognition site is removed and the overhangs will match. The process includes a cyclic temperature program to run an iterative process.
IF we want several parts assembled we can specify the order by designing the flanking 4 nucleotides
How is the destination vector in Golden Gate shuffling processed?
The recognition site is non palindromic and around 4-7 nucleotides. DNA is cleaved outside the recognition site. We use type IIS enzymes that gives a 4-nucleotide overhang. These overhangs can be referred to as fusion sites.
Its important that the fusion sites are facing away from each other in the destination vector because when we then remove the target, the plasmid will not recirculate.
What characterizes type IIS restriction enzymes?
They cleave outside of their recognition site and create 4 nucleotide overhangs. Often used in Golden Gate shuffling.
How is the destination vector and target fragments in Golden Gate shuffling combined?
They are mixed in a reaction with type IIS enzymes and DNA ligase. Cleavage is done inside the fragment so the recognition site is removed and the overhangs will match. The process runs iteratively automatically until all material has been made. It is therefore called a 1 step process unlike BioBrick for example.
How do we order the fragments in Golden Gate shuffling?
By the order of the overhangs
What does 3’exonuclease activity mean?
3’exonuclease activity means it can remove nucleotides from the 3’ end of a DNA strand. The 3’ exonuclease activity can remove overhanging nucleotides from the 3’ end of DNA molecules, creating blunt ends that are easier to ligate into vectors or other DNA constructs.
What is the Golden Gate shuffling library approach?
If we want to create a library for every destination vector with different fragments, we can add them together and have a number of combinations. For example 2 different donor vectors with 3 different fragments in each = 3^2 = 9 different combinations
What is Sequence and Ligation Independent Cloning (SLIC)?
Technique that allows cloning without ligation enzymes, using homology instead and making double stranded DNA
How does Sequence and Ligation Independent Cloning (SLIC) work?
Technique that allows cloning without ligation enzymes. We need the destination vector to be linear. The target fragment is amplified using tail PCR with primers that have approx. 25 nucleotides of homology to the ends of our destination vector! So we have engineered the target fragment to contain the primer from the destination vector!
We use the same flanking region by tail PCR in our PCR product -> the target fragment.
We add T4 DNA polymerase first to the destination vector and the target fragment. The T4 DNA polymerase has 3’exonuclease activity (no dNTPs yet), meaning it can remove nucleotides from the 3’ end of a DNA strand (cut-back reaction). If we expose both the destination vector and the target fragment to the same amount of T4 polymerase, they will eventually create overhangs that match up perfectly to each other based on their homology.
To ensure the T4 polymerase cutting stops we add dCTP. If the vector and fragments have sufficient complementary single stranded 5’ overhands, whey will anneal. Any gaps will be closed in transformation with E.coli
3’exonuclease activity means it can remove nucleotides from the 3’ end of a DNA strand.
What does homology mean?
Very similar DNA, likely originating from the same antcestors
Which DNA assembly methods are based on restriction ligation and which are based on homology?
Restriction ligation: BioBrick and Golden Gate shuffling.
Homology: SLIC, Gibson, CPEC
What is random assembly in Golden Gate shuffling?
Possible if we have several very similar sequences (they share 4 nucleotides in the ends that will serve as out overhangs). We mix all vectors with a destination vector, add E.coli bacteria and ligase. Perform cyclic temperature program and we will have a random library.
What is the main limitation for assembly methods based on restriction ligation?
We cannot have any type IIS sites within the target fragment, because then we will cleave the fragment and actively break it. We also need the 4nucleotide overhang to be non-palindrome and ideally with min 2 nucleotide difference.
