Introduction to Synthetic Promoter Libraries

Modulation of gene expression of i.e. cellular enzyme activities (Solem and Jensen 2002), as well as regulation of transcription are amongst some of the areas where SPLs are currently being used. SPL provides an alternative method for gene regulation compared to older methods, namely those of gene knockouts and strong over expression. These two methods are usually based upon apparent rate limiting steps within metabolic pathways (Jensen and Hammer 1998).

When working with gene regulation, it is important to elucidate where expression levels are optimal for the given gene being worked on. Under these specifications it is essential to be able to have slight increments in expressional strength when attempting to optimize gene expression. This can be achieved by the usage of an SPL, where the variability in strengths can be achieved by either randomizing the spacer sequences, namely the 17 bases that reside between the -35 and -10 consensus regions, and/or some of the bases within the consensus regions, being the -35 and -10 regions.

The spacer sequences that surround the consensus regions contribute significantly to the strengths of promoters (Hammer et al. 2006). In our design, we decided to both randomize the spacer sequences as well as two bases in both consensus regions as seen in Figure 1 below. N stands for 25% each of A, C, G and T, while S stands for 50% each of C and G, and W stands for 50% A and T.

Figure 1: An SPL designed on the basis of randomizing both the spacer sequences surrounding the consensus regions (-35 and -10 regions) as well as randomizing two bases within each of the consensus regions is illustrated. N stands for 25% each of A, C, G and T, while S stands for 50% each of C and G, and W stands for 50% A and T.

The point of randomizing both areas is to obtain a promoter library that is not biased towards being strong. This is achieved by giving two bases within each of the consensus regions a 50% chance of being their original bases, ensuring that only 1/16 of all promoters will be strong. This is without taking into consideration the fraction of strong promoters obtainable from the randomized spacer sequences.

As previous studies indicate, consensus regions outside of the -35 and -10 regions seem to contribute very little in terms of altering promoter strengths. Mutating a single nucleotide will not change the promoter strength substantially, however mutating many nucleotides in the spacer sequences surrounding the -35 and -10 regions seem to result in the most significant alterations in promoter strengths. This might be due to the three-dimensional structure that forms from the sequences that are arranged from the randomized spacer sequences (Jensen and Hammer 1998).

When wanting to characterize and/or fine tune BioBrick parts and devices, using promoters that are constrained to already set strengths, has the disadvantage that the promoter might induce gene expression that is either too high or too low for the cell to be viable. This problem is nonexistent when using SPL since the SPL will necessarily give you the allowed upper and lower bounds of gene expression for cell viability. Cells with too strong or too weak promoters will simply never grow colonies.

SPL as a New BioBrick Standard

Strategy for Integrating SPL into the BioBrick Assembly Standard

There are many different ways to integrate an SPL into the BioBrick Standard, and a lot of ideas were considered when creating this RFC. However, in the end a method was chosen based on the fact that it would be least time consuming for teams looking to use SPL, and at the same time, be easy to do. Instead of relying on ligations to successfully insert the SPL onto the BioBrick plasmid backbone, a Polymerase Chain Reaction (PCR) method was designed to not only amplify the backbone but also add the SPL onto the linear BioBrick plasmid backbone at a specific chosen site (see Figure 2). Since most teams will probably have to amplify their backbones during the course of a project, this method will only require a small amount of extra work.

Wanting to optimize gene expression and thereafter choosing a promoter that conforms to the strength that efficiently expresses your gene would be best perceivable if the SPL could be easily added and removed from BioBrick parts and/or devices. That is why the SPL will be inserted by PCR in-between the restriction sites EcoRI and XbaI of the BioBrick prefix. This way it is possible to add a part downstream of the SPL by simply ligating a part into the backbone plasmid containing SPL or by using the 3A-assembly standard. Furthermore it is also possible to move the whole insert into another BioBrick plasmid backbone if needed.

Figure 2: The linear BioBrick plasmid backbone with SPL inserted between the EcoRI and XbaI sites of the BioBrick prefix is illustrated.

The design of the SPL leads to the possibility of illegal restriction sites being present within the randomized spacer sequence. If a given promoter is to be used in further ligations it is vital that the promoter is sequenced first to ensure that it does not contain any recognition sites for EcoRI, XbaI, SpeI or PstI. The presence of these recognition sites could lead to the promoter being cut in a future restriction digest

Primer Design

A PCR MUST be used in order to add the SPL onto the BioBrick plasmid backbone. The following primers for amplification of BioBrick plasmid backbones were used as a starting point for the design of our SPL primers:

1. Primer Suffix-F: 5’-ACTAGTAGCGGCCGCTGCAG-3’
2. Primer Prefix-R: 5’-TCTAGAAGCGGCCGCGAATTC-3’

• Blue – EcoRI
• Green – XbaI
• Red – SpeI
• Turquoise – PstI

1. Primer SPL Suffix-F: 5’-GTTTCTTCACTAGTAGCGGCCGCTGCAG-3’

Figure 3: The primer binding sites on a BioBrick plasmid backbone as well as the final linear plasmid backbone that is generated by the PCR is illustrated.