NextGen Synthesis

7th of September - Joseph Jacobson (MIT) Recordings

• Download link

• Photoelectrochemical synthesis of DNA microarrays
• Programming cells by multiplex genome engineering and accelerated evolution

• Web http://www.nupack.org/partition/new
• Command line http://www.nupack.org/downloads/register

• PCR Thermocycler
• Vortexer
• Gel Electrophoresis Box
• Power Supply 250V

For questions about the homework, please contact Noah Jakimo - njakimo (at) mit.edu

• Theory: Use NuPack to help select 18 bp priming sites that amplify a ~2.25 kb region of pUC19 (NEB) immediately upstream of the plac promoter and downstream of the start of lacZalpha. The resulting amplicon excludes the plac promoter and n-terminus of lacZalpha, which enables you to swap in a gene of interest under the control of a promoter of interest using Gibson Assembly later on in your workflow. Design one pair of oligos that prime optimally and another that prime poorly. Describe the PCR thermocycling program that uses Phusion polymerase (NEB) for these pairs. Crucially, determine annealing temperatures and extension times.
  • Helpful readings:
    • PCR intro
    • Primer Design
  • Nupack task check list:
    • Number of strand species >= 2 for combinations of primers and ~50 200bp regions from pUC19 containing their reverse compliment
    • Sizes of complexes >= 2 to check self-hybridization, hybrid primer dimers and annealing to target
    • Primer and DNA concentrations are in a range suitable for PCR rx mixture
    • Melt range includes several points in Phusion's annealing temperature range

• Experiment: PCR amplification using combinations of optimal/poor priming sites and exo+/- DNA polymerase. Readout with agarose gel and Sanger sequencing (GeneWiz).

• Extra Credit: Identify sequences elsewhere on pUC19 that most closely match your primers. Use Nupack to assess the likelihood of off-target amplicons.

• Theory: Recode a fluorescent reporter and use NuPack to help design 200-300 bp IDT gBlocks for building the reporter with Gibson Assembly. Check that your 15-30 bp overlaps do not fold into undesirable secondary structure during the isothermal reaction.
  • Helpful readings:
    • Codon Table
    • Gibson Assembly
    • Linear backbone usage

• Experiment: Assemble and amplify gene to readout on gel.

• Extra Credit: Assemble into linearized pUC19 with a promoter, RBS and terminator to express in E coli or extract.

To pass this class, complete theory assignment from homework 1 and 2 - experiments are optional (but recommended).

• If reading the pUC19 genbank text file feels awkward, then please consider downloading a plasmid viewer. SnapGene is used widely and is free.
• Look up or ask about the function of any annotated regions that are unfamiliar to you.
• Instead of selecting primers from the suggested 50 bp window downstream and upstream of lacZa and its promoter, respectively, please select from a broader 200 bp region.
• For part 1 of the homework: If you are having trouble analyzing primers in Nupack or would like to use a more intuitive tool to evaluate primers, then try out IDT's OligoAnalyzer.
• A passing grade on homework part 1 is earned by identifying:
  • One primer pair that you expect to amplify the desired product under broad annealing temperature conditions (50-72C)
  • One primer pair that you expect to be sensitive to annealing temperature and/or allows for extension on undesirable templates
  • An explanation for each selection that includes the melting temperature (Tm) or free energy (delta G) of intra- and homo/hetero inter- strand hybridization
• For part 2 of the homework: When codon-optimizing and parsing a fluorescent protein gene for ideal Gibson overlaps, consider using other appropriate tools (CodonOpt, UNAFold) from IDT.
• A passing grade on homework part 2 is earned by identifying:
  • An E. coli optimized coding sequence for the gene
  • A partition of the coding sequence into 200-300 bp with 15-30bp overlaps
  • An explanation for selected overlap sequences that includes their melting temperature (Tm) or free energy (delta G)
• Note, such overlaps will likely be sufficient for overlap extension PCR, which requires fewer enzymes than Gibson assembly. For the experimental portion, I encourage you to also try assembling your fragments with a similar protocol to this one.