Week 4: Next Generation Synthesis

Summary

The latest game-changer in synthetic biology comes in the form of next-generation gene synthesis, which delivers highly accurate constructs at significantly lower prices and in far greater numbers than has been possible to date.

In the class, taught by Prof. Joe Jacobson, co-founder of Gen9, we discussed the importance of the ability to synthesize a billion base pairs and how this is made possible through a manufacturing revolution paralleled to the silicon chip industry.

Fig. 1: Prof. Joe Jacobson's student Lisa Nip explaining linearization.

Homework Assignment #1: Primer design to linearize plasmid backbone.

Theory

Software needed for the homework assignment: NuPack

Plasmid Map
Fig. 2: Plasmid map for pUC19. In yellow is the designed amplicon.
PRIMER 1:

Scanned 18bp segments, at 4nt step resolution for 5 strand species.

FWD 1: GCCTGGGGTGCCTAATGA
FWD 2: GGGGTGCCTAATGAGTGA
FWD 3: TGCCTAATGAGTGAGCTA
FWD 4: TAATGAGTGAGCTAACTC
FWD 5: GAGTGAGCTAACTCACAT

Specified max complex size at 2 strands (for possible dimers).

Parameters set by Phusion polymerase, at primer concentration of 0.5µM and several annealing temps ranging between 45-72⁰C (5 temps, with 7⁰C increments), assuming TA is 3-5⁰C below TM. You can see the results in Fig. 3.

Fig. 3: Results: (left to right) Equilibruium concentrations histogram, Melt profile (fraction of bases unpaired), and Ensemble pair fractions. .

PRIMER 2:

Scanned 18bp segments, 4nt step res for 5 strand species. (Reverse complement to original sequence.)

REV 1: ATGACCATGATTACGCCA
REV 2: CCATGATTACGCCAAGCT
REV 3: GATTACGCCAAGCTTGCA
REV 4: ACGCCAAGCTTGCATGCC
REV 5: CAAGCTTGCATGCCTGCA

Specified max complex size at 2 strands (for possible dimers).

Parameters set by Phusion polymerase, at primer concentration of 0.5µM and several annealing temps ranging between 45-72⁰C (5 temps, with 7⁰C increments), assuming TA is 3-5⁰C below TM. Results in Fig. 4.

Fig. 4: Results: (left to right) Equilibruium concentrations histogram, Melt profile, and Ensemble pair fractions.
GOOD PRIMER SET:

FWD 2: GGGGTGCCTAATGAGTGA
REV 2: CCATGATTACGCCAAGCT

Similar GC%, and melting temps. No instances of self-annealing or primer dimers between them. Produces a 2.6Kbp amplicon (a little on the large side, target 2.3Kbp)

PCR @ Annealing Temp: 55⁰C
PCR @Extension Time (@ Phusion 15 sec/kb recommendation): 40 sec

BAD PRIMER SET:

FWD 5: GAGTGAGCTAACTCACAT
REV 4: ACGCCAAGCTTGCATGCC

Both primers have GC% out of the 40-60% range, and respectively too low and too high. Because we have to choose a single annealing temperature to use in PCR cycles, the wide range between their GC%/temps adds to the inefficacy of these primers.

Fig. 5: Dot plot of the secondary structure prediction for 4 primers.

Experiment

TBD

Homework Assignment #2: Build a gene from shorter gene fragments.

Theory

TBD

Experiment

TBD