Week 2 HW: DNA Read and Write and Edit

Part 0: Basics of Gel Electrophoresis

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Part 1: Benchling & In-silico Gel Art

DNA Crawler

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Part 2: Gel Art - Restriction Digests and Gel Electrophoresis

Not possible - sadly I don’t have access to a lab. :/

Part 3: DNA Design Challenge

I chose MerR, a metal-responsive transcriptional regulator, because it directly senses toxic heavy metals (such as mercury) and converts that detection into a clear genetic on/off signal. This fits to my idea developed for HW1, where it’s all about detecting heavy metals in water.

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MerR Amino Acid Sequence

sp|P22853|MERR_BACCE Mercuric resistance operon regulatory protein OS=Bacillus cereus OX=1396 GN=merR1 PE=1 SV=1 MKFRIGELADKCGVNKETIRYYERLGLIPEPERTEKGYRMYSQQTVDRLHFIKRMQELGF TLNEIDKLLGVVDRDEAKCRDMYDFTILKIEDIQRKIEDLKRIERMLMDLKERCPENKDI YECPIIETLMKK

MerR DNA Sequence

ATGATGAAAT TTCGTATTGG TGAATTAGCT GATAAATGTG GTGTTAATAA AGAAACTATT CGTTATTATG AACGTTTAGG TTTAATTCCT GAACCTGAAC GTACTGAAAA AGGTTATCGT ATGTATTCTC AACAAACTGT TGATCGTTTA CATTTTATTA AACGTATGCA AGAATTAGGT TTTACTTTAA ATGAAATTGA TAAATTATTA GGTGTTGTTG ATCGTGATGA AGCTAAATGT CGTGATATGT ATGATTTTAC TATTTTAAAA ATTGAAGATA TTCAACGTAA AATTGAAGAT TTAAAACGTA TTGAACGTAT GTTAATGGAT TTAAAAGAAC GTTGTCCTGA AAATAAAGAT ATTTATGAAT GTCCTATTAT TGAAACTTTA ATGAAAAAAT AA

MerR Optimized Sequence

ATGAAGTTCCGGATTGGCGAGTTGGCTGATAAATGCGGCGTGAACAAGGAGACAATCCGATACTACGAGCGTCTGGGTCTGATACCGGAGCCGGAGCGGACTGAGAAGGGATACCGGATGTATTCCCAACAAACCGTGGACCGCCTGCACTTCATAAAGCGTATGCAAGAACTGGGGTTCACGCTCAACGAAATCGACAAACTGTTGGGTGTGGTTGATCGCGACGAAGCAAAATGTCGTGACATGTATGACTTCACGATACTTAAGATAGAGGACATTCAGCGCAAGATTGAGGATCTGAAGAGAATCGAAAGAATGTTGATGGACCTCAAGGAGCGGTGCCCAGAGAACAAGGACATCTACGAGTGTCCGATTATCGAAACGCTGATGAAGAAG

3.4. You have a sequence! Now what?

There are different ways how to get the proteins. There is cell -dependent and a cell-free option.

In a cell-dependent system, the gene, in my case the MerR-gene is inserted into a plasmid along with a promoter. When this DNA is introduced into a host cell (I optimized my sequence for the E.coli bacteria), the cell should be able to read the inserted DNA sequence and reproduce it.

Part 4: Prepare a Twist DNA Synthesis Order

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What DNA would you want to sequence (e.g., read) and why?

I would want to sequence DNA from water samples. By reading this DNA I could try identify genes related to pollution tolerance or resistance (for example, heavy-metal resistance genes??). This information could help reveal hidden environmental stressors and contamination.

I would choose the second-generation, because i would have to sequence lots and lots of DNA in a fast way.

Input:

Extracted environmental DNA (eDNA) from water samples

Essential preparation steps:

  • DNA extraction from water (often after filtration)
  • Fragmentation of DNA into short pieces
  • Adapter ligation to both ends of fragments
  • PCR amplification to enrich adapter-ligated DNA

Essential sequencing steps and base calling

  • DNA fragments bind to the flow cell surface
  • Clusters of identical DNA fragments are generated (bridge amplification)
  • Fluorescently labeled nucleotides are added one base at a time
  • A camera records the emitted fluorescence after each incorporation
  • The color signals are converted into DNA bases (A, T, C, G) through base calling

Output

  • Short DNA sequences
  • Output files containing the DNA sequence
  • These reads can be analyzed to identify species, genes, and indicators of environmental contamination

DNA Write

I would want to synthesize a genetic sensor circuit for environmental heavy-metal detection working in bacteria.

Specifically, I would synthesize a DNA construct containing the merR gene and its metal-responsive promoter, coupled to a odor-producing reporter gene.

  • The MerR protein acts as a sensor: when a toxic metal is present, MerR changes conformation and activates transcription from its target promoter.

  • This promoter would then drive expression of genes that produce a strong smell.

Rather than synthesizing a whole genome, I would focus on a modular genetic circuit, including:

  • metal-sensing regulatory gene (merR),
  • metal-responsive promoter,
  • reporter module encoding enzymes that generate a bad odor.

This approach allows the circuit to be easily tested, tuned, and reused in different bacterial hosts.

Array-based oligonucleotide synthesis (phosphoramidite chemistry) is ideal because it allows many DNA sequences to be synthesized in parallel and relatively low cost. This is well suited for building modular genetic circuits (promoters, genes, regulatory elements) and rapidly iterating on designs.

Essential steps of the DNA synthesis method

  • Digital design of the DNA sequence (e.g. merR, promoter, reporter genes)
  • Chemical synthesis of short DNA on a silicon chip
  • Collection of synthesized oligos from the chip
  • Assembly of oligos into longer DNA fragments
  • Sequence verification before use

DNA Edit

I would want to edit the DNA of bacteria to enhance environmental sensing, specifically by modifying genes involved in heavy-metal detection and response. For example, I could edit the promoter or coding sequence of the merR gene to increase sensitivity to toxic metals, or introduce regulatory sequences that trigger production of a strong, unpleasant odor when contamination is detected.

The reason for these edits is to create a living environmental sensor that makes heavy metals/hazardous matter in water into an percieviable warning. By fine-tuning bacterial DNA in this way, we can make environmental risks tangible to humans through smell, bridging the gap between invisible pollution and instinctive human perception.

Beyond bacteria, similar editing could be applied to plants or other organisms used in biosensing, to increase their ability to indicate pollution or environmental stress without harming the organism or the ecosystem.

CRISPR

For editing bacterial DNA in my environmental sensor project, I would use CRISPR-Cas9 genome editing because it is precise, efficient, and widely used in bacteria.

CRISPR-Cas9 works as a programmable molecular scissor: a Cas9 nuclease is guided by a short RNA sequence (sgRNA) to a specific DNA target. Cas9 introduces a double-stranded break at the target site. The cell’s repair machinery afterwards fixes the break and giving the possibilty to introduce DNA.

Essential steps

  • Design the guide RNA (sgRNA) to target the DNA sequence I want to edit
  • Assemble the CRISPR-Cas9 system (Cas9 + sgRNA) on a plasmid or deliver as RNA/protein complex
  • Provide a repair template if precise edits are needed (for HDR)
  • Introduce the system into the host cells (e.g., bacteria via transformation or electroporation)
  • Allow cells to repair the DNA break, producing the desired edit
  • Screen and verify edited cells using sequencing

Limitations

  • Efficiency: HDR-based edits can be less efficient than NHEJ
  • Precision: Off-target cuts may occur if sgRNA binds similar sequences
  • Cell-dependence: Requires competent host cells capable of repairing DNA breaks
  • Size limitations: Very large DNA insertions can be more challenging