Week 2 HW: DNA Read, Write & Edit

Part 1: Benchling & In-silico Gel Art

First I created an account on Benchling

Then, I imported Escherichia phage Lambda, complete genome with GenBank ID: J02459.1

Fig 1: Linear map of lambda DNA (LAMCG) imported into Benchling.

Next, I simulated restriction enzyme digestion with the following enzymes

  • EcoRI
  • HindIII
  • BamHI
  • KpnI
  • EcoRV
  • SacI
  • SalI

Fig 2: Simulating restriction enzyme digestion of lambda DNA on Benchling.

I then created the following design pattern by simulating combined restriction enzyme digestions in single, double, and triple enzymes.

Fig 3: Design pattern generated by simulated restriction enzyme digestions of lambda DNA on Benchling

Part 2: Gel Art - Restriction Digests and Gel Electrophoresis

Part 3: DNA Design Challenge

3.1. Choose your protein.

One of my ideas for the final project is designing a biosensor-coupled aroma production system in which a detectable scent serves as the output signal of a genetic circuit. Specifically, the project focuses on engineering the biosynthesis of Citral, an aromatic acyclic monoterpene aldehyde which is the primary component responsible for the characteristic lemon scent of Cymbopogon citratus(Lemongrass)

The protein of interest is Geraniol dehydrogenase (GeDH), an NAD⁺-dependent oxidoreductase that catalyzes the oxidation of Geraniol to Citral.

Why GeDH?

This specific protein was chosen for several reasons:

  1. It directly produces the compound responsible for the desired aromatic output, making it a critical component of the biosensor system.
  2. Structurally, GeDH belongs to the medium-chain dehydrogenase/reductase family and functions in the cytosol without requiring complex post-translational modifications such as glycosylation. This makes it well-suited for heterologous expression.
  3. In a biosensor-coupled system, placing the gene encoding GeDH under a stimulus-responsive promoter ensures that the final citral scent is only produced when the target signal is detected. Being at the end of the metabolic chain, GeDH functions as the most efficient reporter enzyme for scent-based detection.

Using UniProt, I obtained a protein sequence for GeDH from Castellaniella defragrans, a gram-negative, strictly aerobic, motile bacterium.

>sp|H1ZV38|GEOA_CASD6 Geraniol dehydrogenase OS=Castellaniella defragrans (strain DSM 12143 / CCUG 39792 / 65Phen) OX=1437824 GN=geoA PE=1 SV=1
MNDTQDFISAQAAVLRQVGGPLAVEPVRISMPKGDEVLIRIAGVGVCHTDLVCRDGFPVP
LPIVLGHEGSGTVEAVGEQVRTLKPGDRVVLSFNSCGHCGNCHDGHPSNCLQMLPLNFGG
AQRVDGGQVLDGAGHPVQSMFFGQSSFGTHAVAREINAVKVGDDLPLELLGPLGCGIQTG
AGAAINSLGIGPGQSLAIFGGGGVGLSALLGARAVGADRVVVIEPNAARRALALELGASH
ALDPHAEGDLVAAIKAATGGGATHSLDTTGLPPVIGSAIACTLPGGTVGMVGLPAPDAPV
PATLLDLLSKSVTLRPITEGDADPQRFIPRMLDFHRAGKFPFDRLITRYRFDQINEALHA
TEKGEAIKPVLVF

3.2. Reverse Translate: Protein (amino acid) sequence to DNA (nucleotide) sequence.

I used NovoPro Reverse Translation tool ➡️ https://www.novoprolabs.com/tools/translate-protein-to-dna to transate the GeoA protein sequence from Castellaniella defragrans

>Reverse Translated sequence of GeoA gene from Castellaniella defragrans
ATGAAYGAYACNCARGAYTTYATHWSNGCNCARGCNGCNGTNYTNMGNCARGTNGGNGGNCCNYTNGCNGTNGARCCNGTNMGNATHWSNATGCCNAARGGNGAYGARGTNYTNATHMGNATHGCNGGNGTNGGNGTNTGYCAYACNGAYYTNGTNTGYMGNGAYGGNTTYCCNGTNCCNYTNCCNATHGTNYTNGGNCAYGARGGNWSNGGNACNGTNGARGCNGTNGGNGARCARGTNMGNACNYTNAARCCNGGNGAYMGNGTNGTNYTNWSNTTYAAYWSNTGYGGNCAYTGYGGNAAYTGYCAYGAYGGNCAYCCNWSNAAYTGYYTNCARATGYTNCCNYTNAAYTTYGGNGGNGCNCARMGNGTNGAYGGNGGNCARGTNYTNGAYGGNGCNGGNCAYCCNGTNCARWSNATGTTYTTYGGNCARWSNWSNTTYGGNACNCAYGCNGTNGCNMGNGARATHAAYGCNGTNAARGTNGGNGAYGAYYTNCCNYTNGARYTNYTNGGNCCNYTNGGNTGYGGNATHCARACNGGNGCNGGNGCNGCNATHAAYWSNYTNGGNATHGGNCCNGGNCARWSNYTNGCNATHTTYGGNGGNGGNGGNGTNGGNYTNWSNGCNYTNYTNGGNGCNMGNGCNGTNGGNGCNGAYMGNGTNGTNGTNATHGARCCNAAYGCNGCNMGNMGNGCNYTNGCNYTNGARYTNGGNGCNWSNCAYGCNYTNGAYCCNCAYGCNGARGGNGAYYTNGTNGCNGCNATHAARGCNGCNACNGGNGGNGGNGCNACNCAYWSNYTNGAYACNACNGGNYTNCCNCCNGTNATHGGNWSNGCNATHGCNTGYACNYTNCCNGGNGGNACNGTNGGNATGGTNGGNYTNCCNGCNCCNGAYGCNCCNGTNCCNGCNACNYTNYTNGAYYTNYTNWSNAARWSNGTNACNYTNMGNCCNATHACNGARGGNGAYGCNGAYCCNCARMGNTTYATHCCNMGNATGYTNGAYTTYCAYMGNGCNGGNAARTTYCCNTTYGAYMGNYTNATHACNMGNTAYMGNTTYGAYCARATHAAYGARGCNYTNCAYGCNACNGARAARGGNGARGCNATHAARCCNGTNYTNGTNTTY

3.3. Codon optimization

Codon optimization is important to maximize the production of the protein of interest when it is expressed in a host organism. This need arises because the native source organism and the expression host often possess different molecular machinery. Although the genetic code is degenerate, different species have evolved preferences for specific synonymous codons, a phenomenon called codon usage bias. So, codon optimization acts as a molecular “translator” of some sorts.

For this project, codon optimization was performed using VectorBuilder, with E.coli selected as the expression host.

Why E. coli?

  • Rapid growth and high yields - E. coli doubles every 20-30 minutes in cheap media, enabling grams-per-liter protein production for quick prototyping.
  • Simple genetics and expression - Plasmid-based systems (e.g., pET/T7) offer tight inducible control without eukaryotic PTMs
  • Cost-effective and scalable - Low setup costs and established protocols make it ideal for iterative testing
Codon optimized DNA sequence of the GeoA gene
ATGAATGATACGCAGGATTTTATTAGCGCGCAGGCGGCAGTACTGCGTCAGGTGGGCGGCCCGCTGGCGGTCGAACCGGTGCGTATCTCGATGCCGAAAGGCGATGAAGTTCTGATTCGTATTGCCGGCGTGGGCGTGTGTCATACCGATCTGGTGTGTCGCGATGGCTTCCCGGTTCCGCTGCCGATTGTGCTGGGCCATGAAGGCAGCGGCACAGTGGAAGCTGTGGGCGAACAGGTTCGCACCTTAAAACCGGGCGATCGCGTTGTGCTGAGCTTTAATAGCTGCGGCCACTGCGGTAACTGTCACGACGGTCATCCGAGCAATTGCCTGCAAATGCTGCCGCTGAATTTTGGTGGTGCCCAGCGCGTTGATGGTGGACAGGTGTTAGATGGCGCCGGTCATCCGGTGCAGAGCATGTTTTTTGGCCAGTCGAGCTTTGGCACCCATGCGGTGGCGCGCGAAATTAATGCGGTGAAAGTGGGCGATGATCTGCCGCTGGAGTTACTGGGACCGCTGGGCTGCGGTATTCAGACCGGCGCCGGCGCCGCCATTAATTCTCTGGGTATTGGCCCGGGCCAGAGCCTGGCCATTTTTGGCGGCGGCGGCGTTGGTCTGAGCGCCCTGCTGGGTGCGCGCGCCGTGGGCGCGGATCGCGTGGTAGTAATCGAGCCGAACGCGGCGCGTCGTGCGCTGGCCCTGGAACTGGGTGCGAGCCACGCGCTGGATCCGCATGCGGAAGGCGATCTGGTTGCGGCCATTAAAGCCGCGACCGGCGGCGGCGCGACCCATAGCCTGGATACGACCGGTCTGCCGCCGGTTATTGGTAGCGCCATTGCGTGCACCCTGCCGGGCGGCACCGTGGGTATGGTGGGACTGCCGGCGCCTGATGCCCCGGTTCCGGCGACCCTGCTGGATCTGCTGAGCAAGTCAGTAACGCTGCGTCCAATTACCGAAGGCGATGCGGATCCGCAGCGTTTTATTCCGCGCATGCTGGATTTTCATCGTGCCGGCAAATTTCCGTTTGATCGCCTGATTACCCGCTATCGTTTTGATCAGATTAATGAAGCGCTGCACGCCACCGAAAAAGGCGAAGCAATTAAACCGGTGCTGGTGTTTTAA

References

Lüddeke, F., Wülfing, A., Timke, M., Germer, F., Weber, J., Dikfidan, A., Rahnfeld, T., Linder, D., Meyerdierks, A., & Harder, J. (2012). Geraniol and geranial dehydrogenases induced in anaerobic monoterpene degradation by Castellaniella defragrans. Applied and environmental microbiology, 78(7), 2128–2136. https://doi.org/10.1128/AEM.07226-11

3.4. You have a sequence! Now what?

To produce GeDH from the optimized DNA sequence, I plan to use both cell-free and cell-dependent systems. The cell-free method will be done first to confirm that the gene is expressed and a functional protein is produced before moving on to a cell-dependent system.

Cell-free

In this case, the optimized DNA sequence will be added to a reaction mixture containing RNA polymerase, ribosomes, tRNAs, amino acids, and nucleotides. The DNA is transcribed into mRNA by RNA polymerase, and the ribosome reads the mRNA in codons, with each codon corresponding to a specific amino acid, which is delivered by a matching tRNA. The amino acids are liked to form a polypeptide chain, which will eventually fold into the functional GeDH enzyme

Cell-dependent

The optimzed DNA will be inserted into an expression vector and introduced into the host organism, E.coli. Inside the cell, the host transcription machinery produces mRNA from the inserted DNA. Ribosomes then translate the mRNA into protein using codon-anticodon pairing. The protein then folds into its 3D structure and becomes active.

3.5. [Optional] How does it work in nature/biological systems?

Describe how a single gene codes for multiple proteins at the transcriptional level. Try aligning the DNA sequence, the transcribed RNA, and also the resulting translated Protein!!! See example below.

Part 4: Prepare a Twist DNA Synthesis Order

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Benchling Link: https://benchling.com/s/seq-a3hKqpNADiAR4fUfT5Sx?m=slm-uo9AlXvO5VzOp4O9Re83

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Part 5: DNA Read/Write/Edit