Week 6 HW: Genetic Circuits Part I: Assembly Technologies
Assignment: DNA Assembly
1. Phusion DNA polymerase is the core enzyme. It has proofreading (3’→5’ exonuclease) activity, which dramatically reduces mutation rates during amplification compared to standard Taq polymerase. GC buffer optimizes the reaction environment. It contains salts and proprietary additives that help denature difficult templates, particularly those with high GC content, by stabilizing the single-stranded state. dNTPs (deoxynucleotide triphosphates) are the building blocks. The mix provides all four (dATP, dTTP, dCTP, dGTP) at balanced concentrations so the polymerase can extend the new DNA strand. Mg²⁺ ions act as essential cofactors. The polymerase requires magnesium to catalyze the phosphodiester bond formation between nucleotides.
2. Primer length and GC content are the primary factors. Longer primers and those with higher GC content have stronger hydrogen bonding, requiring higher annealing temperatures to prevent mismatched binding. Primer-dimer potential matters. If primers can bind to each other rather than the template, you may need to raise the annealing temperature or redesign the primers. Template complexity influences choice. Repetitive sequences or high GC regions in the template may require a lower annealing temperature to allow primers to access their binding sites.
3. PCR amplifies DNA exponentially using thermal cycling, primers, and a polymerase, starting from a tiny template amount. Restriction digests simply incubate purified plasmid DNA with restriction enzymes at a constant temperature, cutting at specific recognition sequences. PCR products are linear from the start and can include overhangs via primer design. Restriction digests produce linear fragments with defined sticky or blunt ends determined by the enzyme chosen. PCR is preferable when you need to amplify a specific region from a complex genome, when template DNA is scarce, or when you want to add custom sequences (like Gibson overhangs) directly during amplification. Digests are preferable when you already have the target sequence in a plasmid, when you need perfectly defined sticky ends for traditional cloning, or when you want to avoid PCR-induced mutations (though high-fidelity polymerases minimize this concern).
4. Design 20-40 bp overlaps between adjacent fragments. These homologous ends must be perfectly complementary and should have a calculated melting temperature around 50-60°C for efficient annealing during the Gibson reaction. Avoid secondary structures at the overlap regions. Hairpins or strong intramolecular base pairing in the overhangs will prevent proper fragment joining. Ensure no internal restriction sites if you linearized by digest. For PCR products, simply include the overlap sequence in your primer’s 5’ end. Verify purity of both PCR and digested fragments. Contaminants like residual restriction enzymes, salts, or ethanol will inhibit the Gibson master mix’s exonuclease and ligase activities. Gel extraction or column purification is essential.
5. Calcium chloride treatment first prepares the cells. The divalent calcium cations neutralize the negative charges on both the bacterial lipopolysaccharides and the DNA backbone, allowing them to come close together. Heat shock (42°C for 45-60 seconds) creates a thermal imbalance. The sudden temperature increase causes the fluid bacterial membrane to become more disordered and permeable, a state sometimes called “thermal permeabilization.” The DNA passively diffuses in during this permeable window. As the membrane expands and proteins reorganize, small pores or transient disruptions allow the plasmid DNA to enter the cytoplasm. The exact mechanism remains debated, but it is not active transport. Cold incubation (on ice) follows immediately. This re-stabilizes the membrane, trapping the DNA inside before the cells are transferred to warm recovery media to repair damage and express antibiotic resistance genes.
6. Golden Gate Assembly uses Type IIs restriction enzymes, such as BsaI or BsmBI, which cut outside their recognition sequences. This creates variable 4-base overhangs that are not part of the enzyme’s binding site. By designing these overhangs carefully, you can assemble multiple DNA fragments in a single, one-pot reaction. The reaction cycles between 37°C (for cutting) and 16°C (for ligation), typically for 30-60 cycles. Each cycle progressively assembles fragments into the final plasmid while the original fragments are cut again if not yet assembled. This method is highly efficient for assembling 4-10 fragments simultaneously and produces scarless junctions. It is the basis for modular cloning systems like MoClo and Golden Braid.
Assignment: Asimov Kernel
Construct_1
This construct directs the expression and secretion of sfGFP out of the E. coli cell. The OmpA signal peptide guides the growing protein to the Sec translocation machinery in the inner membrane, where it is transported into the periplasm. The signal peptide is then cleaved off, releasing mature sfGFP. Some sfGFP may further leak into the culture medium. When induced with IPTG, cells expressing this construct should show reduced cytoplasmic fluorescence (since GFP is exported) but increased fluorescence in the periplasm and supernatant compared to a control without the signal peptide.
Construct_2
In the absence of inducer, LacI protein binds to pLacI and blocks transcription of GFP. When you add IPTG (a lactose analog), IPTG binds to LacI and causes a conformational change that prevents LacI from binding to the promoter. This relieves repression, and RNA polymerase transcribes GFP. The result: no inducer = no fluorescence; inducer added = fluorescence. In the simulation, adding IPTG at 48 hours did not change GFP expression because the LacI repressor was never produced. Your construct only had pLacI → GFP, with no gene to make LacI protein. Without LacI, pLacI is always active, so IPTG has no effect.
Even if LacI were present, IPTG does not instantly remove bound LacI from the DNA. You would need to wait for cell division to dilute out the existing repressor before seeing increased fluorescence.
Construct_3
The first transcription unit (pTetR → TetR gene) produces the TetR repressor protein constitutively. This TetR protein then binds specifically to the pTet promoter in the second transcription unit (pTet → GFP), blocking RNA polymerase from transcribing the GFP gene. As a result, no green fluorescent protein is produced, and the cells remain dark. When you add the inducer aTc (anhydrous tetracycline) to the medium, aTc enters the cells and binds to TetR, causing a conformational change that prevents TetR from binding to pTet. With TetR no longer blocking the promoter, RNA polymerase can now transcribe GFP, producing fluorescence. Therefore, this construct functions as a repressible system: fluorescence is OFF by default and turns ON only when aTc is added. In the TetR-pTet construct, adding aTc successfully changed protein production because the system was properly designed. The TetR repressor was produced constitutively and actively blocked the pTet promoter, keeping GFP off. When we added aTc, it bound to TetR and prevented it from binding to pTet, relieving repression and allowing GFP expression. This worked because both the repressor (TetR) and its target promoter (pTet) were present, unlike the LacI system where the repressor was missing.