Next generation synthesis

In this lab we used synthesized oligonuclease primers to amplify a PCR product. We then used Gibson Assembly on the PCR amplification to assemble a library of protein variant. For an overview of Gibson assembly see the following video from Bio Labs. We will be changing the color-generating chromophore of the purple Acropora millepora chromoprotein (amilCP) to a variety of orange, pink, and blue mutants.


Transformation efficiency: Efficency of cells taking in extracellular DNA, calculated by dividing the number of successful transformants by the amount of DNA used during a transformation procedure (electropration, heatshock, etc.).

5' to 3' exonuclease activity: "chew back", something

Gibbson assembly: A process used to join overlapping gene fragments into a single molecule. See video above. "First a 5' to 3' exonuclease activity creats single stranged 3' overhangs. The complementary sequences then anneal and DNA polymerase extends the 3' ends and fills in the gaps and DNA ligase seals the "nick". This results in fully-sealed, double stranded DNA."

Oligo, oligonucleotides: a polynucleotide whose molecules contain a relatively small number of nucleotides.

Melting temperature: Temperature at which a primer will attach to the template.

Exonuclease: an enzyme which removes successive nucleotides from the end of a polynucleotide molecule.

Ligase: Enzyme that combines DNA fragments together

Primer: A small strand of RNA or DNA

Pathway: A metabolic pathway is a linked series of chemical reactions occurring within a cell.

Flux balance analysis: something

Chromophore: The part of a molecule that gives it color

Vector & Backbone: "The vector itself is generally a DNA sequence that consists of an insert (transgene) and a larger sequence that serves as the "backbone" of the vector. The purpose of a vector which transfers genetic information to another cell is typically to isolate, multiply, or express the insert in the target cell."

Tube terminology:

Lab notes:

The first thing we did was a digestion where we cut up pUC19 plasmids with the restriction enzyme PvuII to generate the linear blunt-ended backbone fragment. At the same time we amplified two sets of gene fragments via polymerase chain reaction (PCR) to be used as inserts in a Gibbson Assembly (i.e. amplicons as inserts).

Note from the lab: "One set of amplicons copy the region of the amilCP gene that precedes the chromophore, including the transcription promoter and translation ribosome-binding site (RBS). Another set amplicons copy the region that spans 24 basepairs before the chromophore to just beyond the gene's transcription terminators. The latter includes a diversified chromophore-coding segment dictated by mismatches in the PCR primers with respect to the mUAV DNA template. The amplicon sets both include one end that overlaps by 20-22 bases with distinct ends of the large backbone fragment from the pUC19 digest."

After we amplied the gene quantity via PCR we had to purify the DNA products as we did last week with the Zymo kit. Lots of centrifuging.

We measusured and adjusted the DNA concentrations using the Nanodrop spectrophotometer to have the appropriate ration in the Gibson Assembly.

To preform the Gibson we mixed the parts, then incubated the mix. Afterwards we heat shocked the assemblies into chemically competent E. coli cells.

After plating the cells we let them incubate at 37C overnight. The photo on the right shows my plate after 18hrs. You can see a single colony growing, but it unfortunately doesn't express the colors I was hoping for. Something went wrong in the pipetting...

You can see cool results by some of my classmates though:

    This is a link to Priya's site.


Q. What are the Gibson overlap sequences in our DNA assembly design? How is an overlap created between the cut pUC19 and the PCR amplified parts of the amilCP gene? Reference see the assignment lecture slides

A. The image below is taken the reference slides. The orange and the blue tags in the bottom right hand corner show the overlap between pUC19 and the amilCP gene that is part of mUAV. VF2 and VR are also labled with purple on the mUAV plasmid.


Q. What is the melting temperature for each overlap sequence and how do they compare to the incubation temperature for the Gibson assembly reaction? refernce (You can use default settings)

A. Putting the above overlaps into the reference link, biophp I get that pUC19 PvuII LrgEnd1 melts at 53.8C and pUC19 PvuII LrgEnd2 melts at 53C.

These are both higher than the incubation temperature of the Gibson assembly, which is 50C. The anealing temperature (Ta) is slightly less than the melting temperature (Tm) of that sequence.From IDT

One consequence of having Ta too low is that one or both primers will anneal to sequences other than the intended target, because internal single-base mismatches or partial annealing may be tolerated. This can lead to nonspecific PCR amplification and will consequently reduce the yield of the desired product. Conversely, when Ta is too high reaction efficiency may be reduced, because the likelihood of primer annealing is reduced significantly. Optimal annealing temperatures give the highest product yield of the correct amplicon.

Q. What is the purpose of each enzyme in the Gibson assembly mix?

A. From addgene:

The Gibson Cloning Master Mix consists of three different enzymes within a single buffer. Each enzyme has a specific and unique function for the reaction:
    T5 Exonuclease - creates single-strand DNA 3’ overhangs by chewing back from the DNA 5’ end. Complementary DNA fragments can subsequently anneal to each other.
    Phusion DNA Polymerase - incorporates nucleotides to “fill in” the gaps in the annealed DNA fragments.
    Taq DNA Ligase - covalently joins the annealed complementary DNA fragments, removing any nicks and creating a contiguous DNA fragment.

Q. (bonus)Our 50 ul PCR reactions included 0.5 uM of both forward and reverse primer and 200 ng of template DNA in the form of a 2,924 bp plasmid. Assuming perfect doubling of DNA copies with each PCR cycle, how many cycles does it take to use all primers? Recall, primers are extended to form copied products. reference
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Q. (bonus)After the heat shock step in our experiment, how can we select for bacteria that contain only the original mUAV plasmid?
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Q. (bonus)Read the following post and its references: Comment on the likelihood of a colony containing two plasmids from our heat shock into NEB 10-beta cells.
A. something