Week 2

Questions from Professor Jacobson:

  1. The natural mechanism for copying DNA is called polymerase.

    What is the error rate of polymerase?: In DNA synthesis, replicative DNA polymerases have an intrinsic error rate of 1:10⁶.

    How does it compare to the length of the human genome?: Comparing with the intrinsic error rate of the polymerase, it is considered that the length of the human genome is large enough for the intrinsic error rate of the polymerase to produce a load of mutations incompatible with life. cellular correction systems.

    How does biology address this discrepancy?: Biology addresses this discrepancy through systems that incorporate cellular correction enzymes, combine parallel synthesis with selection of correct variants, and organize DNA into manageable modules, a strategy that allows scaling the synthesis and maintenance of genetic information togenomic level.

  2. How many different ways are there to encode (DNA nucleotide code) an average human protein? In practice: While the degeneracy of the genetic code allows an average human protein to be encoded by an astronomically large number of distinct DNA sequences, in the practice of synthetic biology a single optimized sequence is designed and selected. This optimization considers biotechnological criteria such as: the use of host preferred codons, the stability of the mRNA and the ease of synthesis. This sequence is then synthesized using high-precision methods and undergoes error correction to ensure its accuracy.

    What are some reasons why all of these different codes don’t work to encode the protein of interest?:

• Organisms have biases in their codon usage.

• Translation speed decreases with rare codons.

• Secondary structures of DNA/RNA affect folding and expression.

• Some sequences generate degradation sites that shorten the half-life of the mRNA.

• Secondary structures of DNA can interfere with synthesis and assembly.

• Some sequences can affect the co-translational folding of the protein.

• Critical regulatory sequences may be altered.

• Technical difficulties in the synthesis of oligonucleotides.

Questions from Dr. LeProust:

  1. What is the most used method currently for the synthesis of oligos?:

    The most advanced method currently used for oligonucleotide synthesis is solid-phase phosphoramidite chemistry, implemented on a large scale using silicon chips. This miniaturized and parallelized approach allows millions of different oligos to be produced with high quality, uniformity and low cost.

  2. Why is it difficult to make oligos larger than 200 nt by direct synthesis?:

• Limited coupling efficiency.

• The byproducts of each cycle accumulate.

• Incorporation errors tend to be more likely at longer lengths.

• Longer oligos can produce secondary structures that block reactive sites, reducing coupling efficiency.

Historically, synthesizing oligos >200 nt via direct synthesis faced chemical limitations that reduced yield and increased errors. However, Twist Bioscience has developed an in-silicon synthesis platform with improved chemistry, which allows oligos of up to 700 nt to be synthesized with high fidelity and purity.

  1. Why can’t a 2000 bp gene be created by direct oligonucleotide synthesis?: You cannot create a 2000 bp gene by direct oligonucleotide synthesis, but you can produce the short fragments that, after assembly and correction, form the complete gene. However, due to the following factors it cannot be done:

• Low performance of complete product.

• High rate of accumulated errors.

• Technical limitations in solid phase synthesis.

George Church Homework Question:

What are the 10 essential amino acids in all animals and how does this affect your view of the “Lysine Contingency”?:

The 10 essential amino acids in all animals are:

  • Histidine
  • Isoleucine
  • Leucine
  • Lysine
  • Methionine
  • Phenylalanine
  • Threonine
  • Tryptophan
  • Valine *Argina

And this affects his view of the Lysine contingency due to the evolutionary dependence on lysine as an essential amino acid in animals, post-translational modification and epigenetic regulation and the implication in recoded organism designs where lysine could be replaced by non-analogues. natural.