Unit 5

____________ is the process of enzymatically joining two DNA fragments. You can think of it as a “paste” function, where you insert the gene of interest into a plasmid.

After ligation, the next step is to insert the recombinant plasmid vector into the host cell. This is known as ____________

DNA _______ is a protein-based enzyme that uses ATP molecules as an energy source to form a covalent phosphodiester bond – joining a 3’OH group and a 5’PO4 group. It is easy to visualize this “paste” function as the opposite of the restriction enzyme “cut” function.

Remember:
During ligation, we are joining a double stranded insert fragment into a __________ double stranded plasmid.
To attach two linear DNA fragments into one circular plasmid, ______ pairs of DNA ends must be joined.

Setting up a Ligation Reaction:

What do we have in our reaction tube?
1) ________ (digested): backbone & stuffer
2) ________(digested)
3) ________ (enzyme required for ligation)
4) ____ (energy needed)
5) B_____and w_______ (two things needed for all reactions)

DNA is taken up directly by cells. Bacteria that are able to take up DNA are called to be “___________”. Some bacteria are “naturally competent”, for example Bacillus subtilis. This is the primary reason for the spread of antibiotic resistance arising from the exchange of plasmids carrying antibiotic resistance genes

Tranformation can be done in 2 ways:

1) _________ transformation: cells are treated with a chemical such as calcium chloride to facilitate attachment of the plasmid DNA to the competent cell membrane. A heat shock treatment then forms pores on the cell membrane allowing the entry of plasmids into the cells. Upon cooling the pores close – now the the plasmids are securely inside the host cell.


2) Electroporation: uses ______ shock for pore formation.

3) The bacterial cells we used were already competent and we didn't have to use the previous two methods

Typical Transformation Controls (there are 3) (I would write this out):

Common transformation controls are:
For transformation controls, we prepare culture plates with growth medium containing a selection marker antibiotic. This antibiotic kills all bacteria, except for this cells that contain the plasmid which offers antibiotic resistance. This is our SELECTABLE marker. Bacteria cannon take up linear DNA – only circular DNA.

(1) Control for ___________ using undigested plasmid: Competent cells should be able to take in any circular DNA _________. To focus of testing competence, we skip the digestion and ligation steps and simple try to transform the undigested circular plasmid.]

(2) Control for __________ activity: To test for ligase activity, we make a single cut on the plasmid, treat it with ligase to re-seal that cut, and then transform the plasmid. If we see colonies, the transformation worked.

(3) Control for ___________.: To test for background growth, i.e. growth resulting from plasmids which have not been properly digested resulting in some circular plasmids – we plate the digested plasmid without any insert.]

Common methods of cloning (inserting gene of interest into a plasmid):

1) ________ _________: Incorporation of restriction sites on ends of PCR products for cloning.

2) ________ ____________: Incorporation of overlapping sequences on ends of PCR products for cloning .
- advantages: fewer steps, more efficient, scarless cloning, can assemble multiple fragments

Which explanation best describes why you will first perform a restriction digest on pHLsec prior to a Gibson reaction? (Note: all are true statements)

a. Forced cloning requires that the restriction sites in a MCS be compatible with the ends of the insert it will join together to properly orient it.

b. pHLsec is a circular plasmid and must be linearized for the exonucleases in a Gibson reaction, as exonucleases need DNA ends to digest.

c. Cutting pHLsec with BamHI and XhoI will remove a 500bp fragment of DNA that will not re-join to the vector in the Gibson reaction.

Carbenicillin, which is similar to ampicillin, is used as the selection agent for bacteria carrying the pHLsec vector. What gene on the pHLsec vector provides the resistance to carbenicillin?

a. OMCP

b. AmpR

c. CMV enhancer

d.ori

The use of divalent cations to make E. coli cells competent causes the membranes to be fragile. We use which of the following method(s) to keep the cells competent until we have added our completed Gibson Assembly reactions:

Select one or more:

a. Thaw cells on ice

b. Keep cells on ice

c. Whisper soothing sounds

d. Gently stir the cells

Gibson Assembly utilizes 3 enzymes. What order must the enzymes work on the DNA ends in order for the insert and vector to be successfully joined?

a. DNA polymerase, then Exonuclease, then Ligase


b. Ligase, then Exonuclease, then DNA polymerase


c. Exonuclease, then DNA polymerase, then Ligase


d. Order is not important

Generally in a ligation reaction, more ________ (vector/insert) than _______ (vector/insert) is used.

Gibson Assembly Reaction Steps:

1) First, __________ chews back the _' ends of the digested plasmid DNA and insert generating long overhangs

2) Then, overlapping sequences from the _______ and ________ anneal

3) Finally, DNA _________ fills in the gaps and DNA ___________ seals the nicks to complete the reaction