KEY CONCEPTS STANDARD CLONING PART 1

THE BEST SET FOR STANDARD CLONING

Basic features of a plasmid backbone to clone and insert

Plasmid + insert = vector 

3 parts needed

1. Origin of replication

2. Antibiotic resistance (selection marker)

3. Multiple cloning site 

What is the purpose of a MCS?

A marker to check if cloning was successful

Type II Restriction Enzymes

Recognize and cut DNA near the specific recognition site 

What are the steps of cloning with a type II restriction enzyme?

STEPS:  

1. Select gene of interest. Amplify by PCR.  

2. Select plasmid vector. Use restriction enzymes to cut the plasmid and place the insert making a vector. Ligation Reaction.  

3. Place vector into bacteria. Transformation.  

4. Culture bacteria on antibiotic plates.  

5. Collect copies of the gene, verify cloned fragments.  

What kind of cuts make sticky ends versus blunt ends?

Symmetrical cuts will leave behind blunt ends. 

Asymmetrical cuts will leave sticky ends.  

3 steps of Ligation reaction

1. ATP activates AMP. Ligase adenylation.  

2. Ligase transfers AMP to 5’P  

3. 3’OH binds to 5’ P forming phosphodiester bond  

Why is the ligation reaction needed?

The ligation reaction is necessary because the backbone of the DNA is broken when the restriction enzymes cut to make room for the insert.  

Function of phophatase?

Phosphatase prevents vector self-ligation by removing the 5’ phosphates. The removal of the phosphates will leave a nick behind which will later be repaired.  

What are double sticky ends?

Double sticky ends are the result of using two restriction enzymes at the same time which both leave behind sticky ends.  

2 types of bacterial transformation

Chemical and Electroporation

Chemical transformation

The heat shock method

The temperature and time of the heat shock will be specific to the bacteria, it’s important not to overdo it. Overdoing will decrease the efficiency of transformation

Steps of CHEMICAL transformation

1. Mix bacteria cells with ligation mix.  

2. Place on ice for one hour  

3. Heat shock  

4. Place on ice  

5. Add LB, let bacteria recover  

6. After 1hr plate onto LB agar  

Electroporation

The cells are prepared in a slightly different way. It is more efficient, but a machine and a cuvette is needed. Ligation mixture and electrocompetent bacteria are mixed together. The mixture is transferred to a cuvette so that an electrical shock can be given. Add LB and let cells recover. After plate the cells on plate with resistance. 

Steps of Eletcroporation

1. Ligation mix + cells  

2. Cells into cuvette  

3. Cuvette into electroporator  

4. Electric shock given  

5. LB is added  

6. Cells recover  

7. Plate the cells on a plate with resistance 

How does electroporation work?

The shock blows holes in the cell wall of bacteria so that the plasmids can go in and transform the target bacteria. 

How does colony PCR work for verification?

1. Swipe a tiny portion of the colony from the plate where the bacteria are growing.

2. Place in tube that has polymerase, nucleotides, and primers specific to either the insert or the vector so you can amplify what's in the cloning site.

3. After amplification the sequence is run on a gel and verified for signs.   

 

If you use insert specific primers, then the only thing that can amplify is the insert but the size verification via the gel still needs to be done because the PCR may have nonpure parts and that was cloned instead of the desired sequence.  

How do you set up culture transformed bacteria?

To do this grow overnight in a culture with LB broth shaking at 37 C. This will cause bacteria to grow exponentially.  

 

On the next day the overnight culture should look very turbid instead of clear indicating that bacteria have grown.  

Why is it important that bacteria be spun down during plasmid mini prep?

It’s VERY important that the bacteria is spun down, if there are any clumps it will not extract well. This step is for high yield analysis.  

Steps of plasmid mini prep

1. Spin down cells and pour off media  

2. Resuspend cells in buffer 

3. Lyse cells with SDS and NaOH  

4. Neutralize cells  

5. Spin to remove debris  

6. Run supernatant over a column to bind DNA to silica  

7. Wash column with bound DNA 

8. Elute in water and use nanodrop to measure 

The plasmid mini prep is just solid phase extraction before PCR.

Why is finger printing DNA done?

Also known as restriction enzyme digest.  

Used to make sure insert and vector length are as expected.  

Steps of finger printing plasmid DNA (restriction enzyme digest)

1. Extract plasmid DNA

2. Cut plasmid with restriction enzymes tha will cut the plasmid and insert

3. Place fragmented DNA on gel so that the pieces separate by size which plasmids contain the insert and which do not

How does sanger sequencing work?

Uses only a single primer to show a single sequence from the prime site. Include ddNTP that are marked with a different flourore. Which will cause a stop and release fluorescence. 

3 ways to improve cloning efficiency (restriction cloning based process)

1. Alpha Complementation aka blue white screening

2. Chromoprotein screening

3. Negative selection using ccdB toxin

Alpha Complementation aka blue white screening

The plasmid with the cloning piece will have the LacZ prime piece, which is a piece of the enzyme galactosidase that allows a synthetic molecule to make a blue substrate

Doing blue white screening is checking for the LacZ insert

Interpreting blue white screening

No insert: LacZ’ complements defective LacZ in bacteria and produces a blue substrate (if the plates have X-Gal)  

Insert: LacZ’ is interrupted and no functional -Gal made: white colonies 

What are the disadvantages of blue white screening?

• X gal molecule is expensive 

• Plates have to be shielded from light 

Chromoprotein screening

A fluorescent or chromoprotein can be inserted into the MCS  

 

Chromoprotein screening is a quick way to check if your DNA cloning experiment worked by using proteins that make bacteria change color.

• Chromoproteins are proteins that naturally look colored (like pink, purple, blue).

• When the DNA you are cloning is correctly inserted into bacteria, the bacteria will make the chromoprotein.

• This means the bacterial colonies on your plate will show up in that color.

• If there’s no color, it usually means the DNA insert isn’t there (or it didn’t work).

What is the advantage of Chromoprotein screening over blue white?

no substrate needed (X-Gal is expensive) 

ccdB toxin

Control of cell death B  

 

Successful insert cloning interrupts ccdB allowing the colonies to grow 

 

To grow the empty vector, you need a strain that protect against ccdB toxicity by expressing the antitoxin ccdA