Week 6 HW: Genetic Circuits Part 1
Assignment: DNA Assembly
1.What are some components in the Phusion High-Fidelity PCR Master Mix and what is their purpose?
Phusion is an enzyme commonly used for high-precision cloning. Its Master Mix typically contains the following core components:Phusion DNA Polymerase: The core enzyme. It possesses 3’→5’ exonuclease activity (proofreading function), ensuring high fidelity DNA replication with an extremely low error rate. dNTPs (dATP, dTTP, dCTP, dGTP): The “raw materials” for building new DNA strands.Mg²⁺ (usually MgCl₂): A cofactor for the polymerase. It stabilizes primer-template binding and activates the enzyme’s catalytic center. Buffer: Maintains a constant pH and provides suitable ionic strength to ensure enzyme activity.
2.What are some factors that determine primer annealing temperature during PCR?
The primer annealing temperature (Ta) is typically 3-5°C lower than the primer melting temperature (Tm). Influencing factors include:1. Primer length: Usually between 18-30 bp; longer primers generally result in higher Tm. 2.GC content: G-C base pairs have 3 hydrogen bonds, while A-T pairs only have 2. Therefore, higher GC content leads to higher Tm. 3.Salt ion concentration: The concentration of monovalent cations (such as Na+, K+) and divalent cations (Mg2+) in the reaction system neutralizes the negative charge of the DNA backbone; higher concentrations result in higher Tm.4.Mismatch degree: Imperfect matching between primers and templates significantly reduces annealing efficiency and temperature.
3.There are two methods from this class that create linear fragments of DNA: PCR, and restriction enzyme digests. Compare and contrast these two methods, both in terms of protocol as well as when one may be preferable to use over the other.
Reaction Mechanism and Principle:PCR is an in vitro enzymatic amplification process that uses primers to guide polymerase to synthesize new strands de novo on a DNA template. Restriction enzyme digestion, on the other hand, is an enzymatic cleavage process that uses restriction endonucleases to recognize specific sequence sites and “cut” the DNA; it does not involve the synthesis of new DNA.
Product properties and yield: PCR can achieve exponential amplification of target fragments, obtaining a large number of DNA fragments from a very small amount of template; in contrast, restriction enzyme digestion produces fragments in a fixed ratio to the starting material, with no increment.
Design flexibility: PCR has extremely high flexibility. Researchers only need to design corresponding primers based on the target sequence to obtain DNA fragments at almost any position. In contrast, restriction enzyme digestion is strictly limited to the enzyme sites that are naturally present or artificially constructed in the DNA sequence. Without suitable recognition sites, it is impossible to perform cleavage.
Advantages, disadvantages, and accuracy: The advantages of PCR are its speed and high yield, which can quickly obtain a large amount of target DNA. However, since it is artificially synthesized, base mutations may be introduced during the process. Restriction enzyme digestion can completely maintain the accuracy of the original sequence and will not produce new mutations, but its application is highly dependent on the specific sequence background.
4.How can you ensure that the DNA sequences that you have digested and PCR-ed will be appropriate for Gibson cloning?
Designing Overlapping Regions: Adjacent fragments must have 20-40 bp of homologous overlapping sequences. These are typically added to the 5’ end of PCR primers.
End Integrity: PCR products require purification to remove excess primers and single nucleotides; restriction enzyme digestion must ensure clean cuts that conform to the overlap design.
Conflict Elimination: Ensure that the fragments do not contain extremely strong secondary structures that conflict with the Gibson reaction temperature (50°C).
5.How does the plasmid DNA enter the E. coli cells during transformation?
Competent cell treatment: Cells are treated with CaCl₂, which bridges the negatively charged DNA phosphate backbone to the equally negatively charged phospholipid layer of the cell membrane via Ca²⁺ ions.
Heat shock: The mixture is rapidly transferred from 0°C to 42°C. This abrupt temperature change creates transient “thermal pores” on the cell membrane, allowing DNA to enter the cell via an electrochemical gradient.
6.Describe another assembly method in detail (such as Golden Gate Assembly)
Golden Gate Assembly utilizes type II S restriction enzymes (such as BsaI or BsmBI) and T4 DNA ligase in the same reaction system. A characteristic of type II S enzymes is that their cleavage sites are located outside the recognition sites, allowing researchers to fully customize the resulting 4bp sticky ends. Through careful design, these recognition sites are removed after cleavage, preventing further cleavage of the product. This method can precisely assemble multiple fragments (typically more than 10) at once, and it is directional. Because the recognition sites are ultimately eliminated, it is also known as “traceless assembly.”
Assignment: Asimov Kernel
Functional Expectation Based on the logic of the negative feedback loop, it is expected that this system will not reach a steady state but will instead generate continuous periodic oscillations. When the concentration of one protein increases, it will suppress the expression of the next gene, causing the concentration of the next protein to decrease, thereby releasing the suppression on the next gene.
Simulation Results & Data Analysis
The chart shows the distinct alternating oscillation behavior of the three components (LacI, LambdaCI, TetR). There is a fixed phase difference between the peaks of each component, which proves that the delay inhibition logic in the loop is operating normally. The oscillation tends to stabilize after approximately 10 hours, maintaining a constant amplitude and frequency.
Construct 1
Based on the classic Repressilator (with three genes: LacI, LambdaCI, and TetR) circuit, I added an sfGFP expression cassette. The fourth group of components is driven by the pTetR promoter and expresses sfGFP. Since pTetR is inhibited by the TetR protein within the system, the expression of sfGFP should be able to reflect the real-time dynamic state of the oscillator.
It is expected that within the 70-hour simulation period, sfGFP will generate continuous periodic fluctuations in conjunction with LacI, LambdaCI and TetR. The simulation curve shows that the system exhibits distinct oscillatory characteristics in the first 10 hours of the simulation. However, as time progresses, the amplitude of the oscillations gradually diminishes (Damped Oscillation), and eventually reaches a stable steady state (Steady State) approximately 20 hours later.
Construct 2
Unlike the independent report branch of Plan 1, in Plan 2, the coding sequence of sfGFP is directly connected to the end of the repressor protein LacI, forming a single transcription unit: pTetR -> A1 RBS -> LacI -> sfGFP -> Terminator.
Plan 2 did not exhibit attenuation! This indicates that the way of fusing the proteins has a lesser impact on the system’s dynamic equilibrium, thus maintaining a long-term periodic behavior.