Synthetic Biology & DNA Assemb

Cheatsheet Content

### Introduction to Synthetic Biology - **Definition:** Engineering biological systems for new functions. - **Goal:** Design and construct new biological parts, devices, and systems, or re-design existing natural biological systems. - **Key Concepts:** - **Top-down design:** Breaking down complex systems into manageable modules. - **Bottom-up design:** Assembling basic biological parts into more complex systems. ### Artificial Tissue Homeostasis for $\beta$ Cells - **Problem:** - Diabetes Type I: Autoimmune response destroys pancreatic $\beta$ cells. - Goal: Maintain $\beta$ cell population using auto-regulated differentiation of ES cells to counter autoimmune attacks. - **Top-down design example:** - Uses ES cells, intermediate cells, and $\beta$ cells. - Feedback loops (stem cells feedback, committed cells feedback) regulate differentiation and division. - **Initial Design Problem:** $\beta$ cell feedback delay is too long, leading to uncontrolled stem cell differentiation. - **Revised Design:** - Engineer cells to commit to differentiation. - Use commitment state for feedback regulation, rather than waiting for full $\beta$ cell differentiation. - Introduces a "commitment latch" to control cell fate. ### GRO Cell Programming Language - **Purpose:** Simulating cellular behavior and interactions in engineered systems. - **Applications:** E. coli nations, morphogenesis, spots, coupled oscillators. - **Example Simulations:** Show how uncommitted and committed cells behave under different growth and commitment conditions. - **Revised GRO Design:** Incorporates stochastic elements (e.g., `rand(10000) > commit_coin_flip`) to stabilize the system, preventing overcommitment of cells. ### Gene Network Architecture - **Components:** - **Uncommitted Population Control:** Oscillator (A0, R1, R2), Rec1, GAF. - **Committed Population Control:** Rec2, I2. - **Commitment Latch Module:** AND gate (A2, R3, R4), Commitment Latch (R5, R6, R7). - **Differentiation Module:** Ngn3, Pdx1, Gata4, AFP. - **Logic Gate Example: Commitment Latch:** - **Gate-Level Logic:** Uses NOR gates (R6, R7) and an inverter (R5) to create a bistable switch. - **Genetic-Level Logic:** Implements the latch using promoters (P6, P7) and regulatory elements. ### DNA Assembly Methods - **Overview:** Various techniques to join DNA fragments to create new genetic constructs. - **Key Principles of Joining DNA:** - Chemical addition - Ligation - Polymerization - Recombination #### Traditional Ligation Cloning (Restriction Enzyme Cloning) - **Principle:** Utilizes restriction enzymes to cut DNA at specific recognition sites, creating 'sticky' or 'blunt' ends, which are then joined by DNA ligase. - **Process:** 1. **DNA Preparation:** Isolate or synthesize the DNA insert fragment and the plasmid vector. 2. **Restriction Digestion:** Treat both the insert and the vector with one or two compatible restriction enzymes to generate complementary overhangs (sticky ends) or blunt ends. For example, using *EcoRI* on both insert and vector will create matching GAATTC/CTTAAG overhangs. 3. **Ligation:** Mix the digested insert and vector in the presence of T4 DNA ligase and ATP. The ligase forms phosphodiester bonds between the DNA backbones, covalently joining the fragments. 4. **Transformation:** Introduce the ligated plasmid into competent bacterial cells (e.g., *E. coli*) for replication and selection. - **Example:** To insert a gene into a plasmid using *EcoRI* and *BamHI*: First, a gene of interest is amplified by PCR, and *EcoRI* and *BamHI* restriction enzyme sites are engineered into its ends. Separately, a target plasmid vector (e.g., pUC19) is digested with the same *EcoRI* and *BamHI* enzymes, which linearizes the plasmid and creates complementary sticky ends. The digested gene fragment and the linearized plasmid are then mixed together with T4 DNA ligase and ATP. The ligase joins the sticky ends, forming a recombinant plasmid. Finally, this recombinant plasmid is introduced into competent *E. coli* cells via transformation for replication and selection. - **Advantages:** Established, inexpensive for single fragment insertions. - **Disadvantages:** Requires compatible restriction sites, can leave 'scars' (restriction sites) at junctions, difficult for multi-fragment assembly. - **Multiple Cloning Site (MCS):** A short segment within a plasmid containing many unique restriction enzyme recognition sites, facilitating the insertion of foreign DNA. - **Important Note (Key Concept from Lecture):** PCR products, especially from proofreading polymerases like Phusion, typically have blunt ends. Ligation of blunt ends requires phosphorylated primers. Without phosphorylation, ligation cannot occur. To get sticky ends from PCR, a digestion step is often needed. Non-proofreading polymerases can add a single A nucleotide (TA cloning), but this is non-directional. #### DNA Assembly from Reusable Parts (BioBricks/Standardized Parts) - **Concept:** Standardized biological parts (promoters, ribosome binding sites (RBS), coding sequences (ORFs), terminators) are designed with common flanking restriction sites, allowing them to be assembled like LEGO bricks. - **Example:** In the BioBrick standard, a promoter part (flanked by *EcoRI* and *XbaI* sites) can be joined to an RBS part (flanked by *XbaI* and *SpeI* sites). By digesting the promoter with *XbaI* and the RBS with *SpeI*, and then ligating them, a composite part is formed. This composite part can then be further combined with other standardized parts using compatible restriction sites. - **Benefits:** Modularity, reusability, facilitates sharing and collaboration, reduced design complexity for synthetic biological circuits. - **Disadvantages:** Can still leave 'scars' (e.g., *SpeI* + *XbaI* can only be ligated if one is cut first, leaving a scar) and limitations in assembly order. - **Important Note (Key Concept from Lecture):** BioBricks represent an early attempt at standardization in synthetic biology, aiming for interchangeable parts. However, the 'scar' sequences left after assembly were a limitation for some applications. #### Gibson Assembly (Gibson Isothermal Assembly) - **Principle:** A one-pot, isothermal reaction that joins multiple DNA fragments based on short homologous overlaps (typically 20-40 bp) with high efficiency. - **Enzymes Involved:** - **T5 Exonuclease:** Chews back DNA from the 5' ends, creating single-stranded overhangs. - **Phusion DNA Polymerase:** Fills in the gaps in the annealed fragments. - **Taq DNA Ligase:** Seals the nicks in the DNA backbone. - **Process:** 1. **Fragment Preparation:** Design PCR primers to amplify DNA fragments, incorporating ~20-40 bp of homologous sequence at the ends that overlap with adjacent fragments or the vector. 2. **Exonuclease Chew-back:** The T5 exonuclease acts on the 5' ends of the DNA fragments, exposing the homologous single-stranded regions. 3. **Annealing:** The complementary single-stranded overhangs from adjacent fragments (or fragment and vector) anneal to each other. 4. **Gap Filling & Ligation:** The DNA polymerase extends the 3' ends, filling in any gaps. Finally, DNA ligase seals the remaining nicks to form a covalently closed DNA molecule. - **Example:** To create a plasmid containing a promoter, a gene, and a terminator, all three fragments are PCR-amplified with overlapping sequences designed into their ends. The plasmid backbone is also linearized. These four components (linearized vector + promoter + gene + terminator) are then mixed together with the Gibson Master Mix (containing the exonuclease, polymerase, and ligase). The enzymes work concurrently to chew back, anneal, fill gaps, and ligate the fragments into a single circular plasmid. - **Key Concept (from Lecture):** Gibson assembly includes a *chew-back step* to generate the required long overhangs (typically 20-40 bp), allowing direct use of PCR products without prior digestion. This method is highly efficient for *multi-fragment assembly* in a single step and results in *seamless (scarless) joins*. - **Advantages:** Seamless (scarless) assembly, highly efficient for multiple fragments (up to 10-15), no restriction enzymes needed, single-step reaction. - **Disadvantages:** Requires longer homologous regions (primers can be expensive), sensitive to primer design errors, products generally need sequencing verification due to potential PCR errors. #### Golden Gate Assembly (MoClo - Modular Cloning) - **Principle:** Leverages Type IIs restriction enzymes (e.g., *BsaI*, *BsmBI*, *BpiI*) that cut DNA outside of their recognition sequence. This allows for the creation of custom, non-palindromic overhangs (fusion sites) that are specific to the desired assembly order. - **Key Feature:** The restriction enzyme recognition site is removed during the cleavage and ligation process, preventing re-cutting of the assembled product. - **Process:** 1. **Fragment Preparation:** Design DNA fragments (e.g., promoter, gene, terminator) with specific Type IIs restriction sites and unique 4 bp overhangs (fusion sites) at their ends. 2. **One-Pot Digestion & Ligation:** Mix all fragments, a linearized destination vector, a Type IIs restriction enzyme, and T4 DNA ligase in a single reaction. 3. **Cyclic Reaction:** The enzyme cuts the fragments, generating specific overhangs. The ligase then joins compatible overhangs. Once ligated, the restriction site is destroyed, making the assembly irreversible and highly efficient. - **Hierarchical Assembly (Modular Cloning - MoClo):** - **Level 0 (Basic Parts):** Individual genetic elements (promoters, RBS, genes, terminators) are cloned into entry vectors. - **Level 1 (Transcriptional Units):** Multiple Level 0 parts are assembled into a transcriptional unit (e.g., promoter-RBS-gene-terminator) in a Level 1 destination vector. - **Level 2 (Multigene Constructs):** Multiple Level 1 transcriptional units are assembled into a multigene construct (e.g., an operon or a pathway) in a Level 2 destination vector. - **Example:** To assemble an expression cassette containing a promoter, an RBS, a GFP gene, and a terminator, each of these parts is designed with flanking *BsaI* restriction sites that generate unique 4 bp overhangs. All four parts, along with a destination plasmid vector, are added to a single tube containing *BsaI* enzyme and T4 DNA ligase. The *BsaI* cuts the parts, and the ligase joins them in the correct order based on the complementary overhangs, forming the complete expression cassette in the destination plasmid. - **Key Concepts (from Lecture):** Golden Gate assembly requires *Type IIs restriction enzymes* and allows for *scarless assembly* and *hierarchical construction*. The overhangs are independent of the recognition sequences, enabling the assembly of multiple sequences with different overhangs together with the same recognition sequence in a one-pot, *one-step reaction*. It offers precise control over the order of assembled fragments. - **Advantages:** Scarless assembly, highly efficient and accurate, hierarchical design enables modularity and complex multigene constructs, no need for gel purification after digestion. - **Disadvantages:** Requires careful design of fusion sites and Type IIs sites, can be more complex to design initially. #### Gateway Assembly (Gateway Recombination Cloning) - **Principle:** Employs site-specific recombination enzymes (integrases) from bacteriophage lambda to transfer DNA fragments between specially designed plasmids. Based on the `att` sites (`attP`, `attB`, `attL`, `attR`). - **Enzymes Involved:** - **BP Clonase:** Catalyzes recombination between `attB` and `attP` sites, creating `attL` and `attR` sites. Used for creating Entry Clones. - **LR Clonase:** Catalyzes recombination between `attL` and `attR` sites, creating `attB` and `attP` sites. Used for creating Expression Clones. - **Process:** 1. **BP Reaction (Entry Clone Creation):** Your DNA fragment of interest (e.g., a PCR product with engineered `attB` sites) is recombined with a "Donor vector" (containing `attP` sites) using BP Clonase. This creates an "Entry Clone" containing your fragment flanked by `attL` sites. 2. **LR Reaction (Expression Clone Creation):** The Entry Clone (with `attL` sites) is then recombined with a "Destination vector" (containing `attR` sites and often a selectable marker like `ccdB` toxicity gene) using LR Clonase. This transfers your fragment into the Destination vector, creating an "Expression Clone" flanked by `attB` sites. The `ccdB` gene provides strong negative selection against non-recombinant vectors. - **Example:** To express a protein in mammalian cells: First, a gene of interest is amplified by PCR, adding `attB1` and `attB2` recombination sites to its ends. This `attB`-flanked gene is then combined with a "pDONR" Donor vector (which contains `attP` sites) in a BP recombination reaction using BP Clonase. This creates an "Entry Clone," where the gene is now flanked by `attL1` and `attL2` sites. Next, this Entry Clone is combined with a "pDEST" mammalian expression vector (containing `attR1` and `attR2` sites, often with a `ccdB` counter-selection gene) in an LR recombination reaction using LR Clonase. This transfers the gene from the Entry Clone into the Destination vector, resulting in an "Expression Clone" ready for protein expression in mammalian cells. - **Key Concepts (from Lecture):** Gateway uses *site-specific recombination* and does not require any overhangs in the starting products. It's excellent for transferring genes between different vector backbones and expression systems interchangeably. The reaction is reversible, but the use of the `ccdB` gene provides a powerful *counter-selection* mechanism to ensure only successful recombinants survive. - **Advantages:** Highly efficient and directional cloning, robust for high-throughp`ut applications, allows easy transfer of genes between different expression systems (e.g., bacterial, yeast, mammalian) by using different Destination vectors, scarless. It enables quick insertion into different destination vectors as long as they have the same recognition sequences for recombination. - **Disadvantages:** Requires specialized Gateway vectors and enzymes, a multi-step process for initial entry clone creation. Multi-way Gateway cloning is possible but generally limited to less than or equal to four fragments due to the scarcity of unique `att` sequences. ### Creation of Synthetic Genomes - **Genome Reconstruction:** Synthesizing an entire genome from scratch based on a known sequence. - **Example: Mycoplasma genitalium Genome Synthesis:** - **Watermarks:** Synthetic DNA includes hidden codes (watermarks) for identification. - **Five-stage assembly:** Large fragments (up to 580kb) are assembled hierarchically from smaller cassettes. - **In vitro recombination:** Cassettes are assembled using methods like Gibson assembly. - **Example: Mycoplasma mycoides JCVI-syn1.0:** - First self-replicating synthetic bacterial cell. - Genome was synthesized and transplanted into a recipient cell. - **The Vision for Synthetic Cells:** - **Design-Build-Test-Learn Cycle:** Iterative process of designing, building, testing, and learning from synthetic biological systems. - **JCVI-syn3.0:** A minimal synthetic cell with a significantly reduced genome (531,490 bp) compared to JCVI-syn1.0 (1,078,809 bp). - **Ethical and Technical Challenges:** - **Technical:** Complexity of large-scale DNA synthesis, potential for errors, ensuring functionality. - **Ethical:** "Creating new life," biosafety concerns, societal implications.