Experiments

Experiments

For the purposes of our project, our primary goal was to demonstrate the feasibility of expressing pterostilbene in E. coli in a reliable manner for the purposes of treating Alzheimer’s disease (AD). Detailed below are the procedures we utilised to test the production of pterostilbene in E. coli using a synthetic operon system.

Flowchart of Steps

To successfully clone multiple genes into a single plasmid using type IIS assembly, our team followed a sequence of discrete steps (see figure 1).

 

 

Construct Parts

To create the synthetic operon, system eight parts were needed (excluding the backbone). The first part is a promoter sequence. From work done by Heo et al.(2017) and Yan et al. (2021), it was decided to use strong promoters to transcribe the operon mRNA. To allow for control over the expression system, the BBa_J23100 promoter sequence was fused to the lac operator sequence BBa_K1624002 as well as the ribosomal binding site BBa_B0030, forming the part BBa_K4388013. Using a promoter-replacing linker and a terminator-replacing linker, the sequence was arranged as displayed in Figure 2.

 

Based off of dry-lab modeling, the plasmid used for final expression of all four genes was the pJUMP49-2A JUMP plasmid available on the iGEM 2022 registry plate kit 1 in well number 6A. For level 1 assembly, pJUMP29-1A/B/C/D were chosen in iGEM 2022 distribution kit plate 1 wells 2A, 2C, 2E and 2G, respectively.

For more details on how to use JUMP plasmids, please refer to the guide we have written below:

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Cloning Parts into Level 1 Plasmids

For all cloning experiments we used the DH5-ɑ strain of E. coli cells. Our team cloned sequences synthesized from IDT into pJUMP29-1A/B/C/D plasmids to form four level 1 parts (see figure 2). Notably, these parts are not transcriptional units, only the pJUMP29-1A level 1 plasmid will have a promoter and only the pJUMP29-1D level 1 plasmid will have a terminator (see table 1). The restriction enzyme used was BsaI. Note that the plates used for level 1 assembly should be Kanamycin as this is the antibiotic resistance gene present in level 1 JUMP plasmids.

Table 1. Depicting plasmid backbones for level 1 assembly alongside level 0 part inserts.

The following protocol was used to digest and ligate the parts together:

Cloning Parts into Level 2 Plasmids

Cloning into a level 2 plasmid requires the BsmBI enzyme and ligated pJUMP29-1A/B/C/D plasmids (see table 1). Otherwise the protocol for ligation follows a similar methodology to the cloning into level 1 parts. Note that the plates used for level 1 assembly should be kanamycin as this is the antibiotic resistance gene present in level 2 JUMP plasmids.

Because level 1 to level 2 JUMP assembly makes use of the assembly of plasmids compared to linear sequences (as one done in the level 1 assembly), there is an increased risk of co-transformation by which a level 1 plasmid can co-transform with a level 2 JUMP plasmid and use the spectinomycin resistance gene of the level 2 plasmid to keep it safe within an E. coli cell. Since this can alter concentrations of enzymes it is important to make sure that this does not occur. Colony PCR can be used to determine plasmid copy number easily. For the purposes of our parts, P1-P2 primers were deemed optimal for JUMP assembly. Below is presented the colony-PCR protocol.

Culturing

For the culturing of our E. coli, we used a protocol described by Heo et al. (2017). As there is a long period of time needed for bacterial culturing (24 hours) it was decided that a rich medium such as terrific broth would be required to ensure bacteria can survive for long. The methodology itself remains relatively simple, one trial with E. coli without the level 2 plasmid and one with the four genes expressed. Detailed below is our culturing protocol for producing pterostilbene.

HPLC-UV

Based on multiple research papers, it was determined that HPLC-UV was the optimal method for measuring the yield of pterostilbene (Heo et al., 2017; Wang et al., 2015; Yan et al., 2021). After 24 hours, cell lysate containing pterostilbene is run through an HPLC-UV system. Details of the HPLC-UV protocol are detailed below.