ICSynBio:Methods Overview

Overview of Techniques

A combination of Golden Gate Assembly and Gibson’s Assembly techniques were utilised for construction of the toggle switch.

Golden Gate assembly is a cloning method which exploits the enzymatic properties of Type IIs restriction endonucleases and T4 DNA ligase to join multiple DNA fragments into a single DNA part, simultaneously and unidirectionally. Type II restriction enzymes recognise 6bp nonpalindromic sequences and cleave DNAs outside of these recognition sequences, resulting in unique 4bp overhangs (single stranded DNA). These overhangs are used as the fusion sites, and directionality is maintained as a result of 3′ fusion site of the upstream part complementing the 5′ fusion site of the following. The resulting overhangs on adjacent DNA fragments anneal, and are joined together using T4 DNA ligase to form a transcriptional unit (TU). A typical assembly produces a transcription unit with four parts (promoter, ribosome binding site, coding sequence, and terminator) assembled into a backbone – usually a DVK (kanamycin-resistance) destination vector or DVA (ampicillin resistance) destination vector).

Gibson assembly (Figure 13) allows the joining of multiple DNA fragments (up to 15 pieces) in a single, isothermal reaction using exonucleases, DNA polymerase, and DNA ligase. Adjacent DNA fragments must overlap by 20-40bp in order to ensure successful assembly. The exonuclease cleaves DNA from the 5' end, exposing complimentary ssDNA. Resulting ssDNA sections on neighbouring DNA fragments can anneal, with the DNA polymerase incorporating nucleotides to fill spaces, which are subsequently covalently joined by DNA ligase.

In Silico Assembly

Due to the unpredictable nature of molecular cloning techniques, two methods were pursued in tandem to increase probability of success.

Method 1: Golden Gate Only Assembly

Figure 14 shows an overview of the Golden Gate Assembly method. DNA parts are placed into destination vectors (with 4 parts per backbone) to form four transcription units (TUs) - GGO_A, GGO_B, GGO_C, and GGO_D. Subsequently these four plasmids are assembled into backbone of DVK_AH to construct the whole toggle switch plasmid GGO_E. Each circle represents unique complementary overhanging sequences between adjacent parts.

Step 1: Spacer creation Four new spacers were created manually. They were necessary for the correct ligation of consecutive parts. A specific sequence for the overhangs on either side of the spacer can be generated, so spacers (Figure 15) can be used to link two parts that do not have complementary overhangs together.

Step 2: Construction of GGO_A, GGO_B, GGO_C and GGO_D All constituent plasmids were created by in silico assembly of BioBricks. A schematic of each plasmid was made, showing their backbone and constituent parts. (Figure 16)

These parts were assembled in silico via simulated Golden Gate Assembly on Benchling. The finished products, Plasmids GGO_A to GGO_D are shown in Figure 17.

Step 3: Construction of GGO_E GGO_E (Figure 18) is the final toggle switch construct. To ensure successful directional assembly, adjacent plasmids have unique complementary bases present on their overhangs. (Figure 19).

Method 2: Golden Gate and Gibson Assembly

Figure 20 shows an overview of the Golden Gate and Gibson assembly method. DNA parts are placed into two destination vectors (with 4 parts per backbone) to form two transcription units (TUs) using Golden Gate assembly - GGG_A, and GGG_B. Subsequently these are used to form GGO_C and GGO_D, which in turn make up GGO_E which contains the whole toggle switch construct.

Step 1: Construction of GGG_A and GGG_B (via Golden Gate Assembly) Constituent parts (Figure 21) were assembled via simulated in silico Golden Gate Assembly to construct plasmids GGO_B and GGO_A (Figure 22).

Step 2: Construction of GGG_C and GGG_D (via Gibson Assembly) Subsequently, virtual Gibson’s assembly was used to linear fragments were created from GGG_A and GGG_B using PCR, which formed plasmids GGG_C and GGG_D (Figure 23). The primers used for this reaction (Appendix C) was designed by Benchling according to strict parameter (Appendix C)

Step 3: Construction of GGG_E (via Golden Gate Assembly) GGG_E (Figure 24) is the final toggle switch construct. To ensure successful directional assembly, adjacent plasmids have unique complementary bases present on their overhangs. (Figure 25).