Procedure

There were two basic goals for this project:

1. Engineer a temperate phage (specifically λ) to synthesize KillerRed in E. coli
2. Demonstrate KillerRed's ability to kill E. coli

Phage Construct

The first goal was achieved using a gt11 lambda kit by cloning in the KillerRed and mRFP1 coding sequences into a β-galactosidase sequence, which results in a β-gal fusion protein to help with solubility. This also includes a single wild-type lac promoter, which responds to IPTG induction. Once clones were identified and isolated, the phages were then proliferated using a strain of bacteria that favors the lytic cycle. An expression strain was used to detect the presence of the fluorescent proteins to help determine if the proteins were present. mRFP1 and KillerRed fluorescence signals were detectable but KillerRed's fluorescence was barely above the autofluorescence of the media. This was repeated several times to determine the reason for KillerRed's low expression. λ lysogen strains were isolated after infection of a high-frequency-of-lysogeny (hfl) strain, which was used to help characterize the effectiveness of the killing by KillerRed. This construct is single copy, as is the nature of λ prophage, which contributed to the low level of expression. There was not enough protein for us to see killing by KillerRed. Ultimately, we were able to develop a lysogen that contains the gene for KillerRed but were having difficulty with expressing detectable amounts of protein via fluorescence.

In parallel, a plasmid construct (using pSB1A2) was made for easy expression and to prove that KillerRed is able to kill bacteria. The plasmid construct consisted of a wild type lac promoter, like the phage and the 100% efficiency RBS standard as shown in Figure 1. Both KillerRed and mRFP1 were cloned so that the mRFP1 acted as the negative control. Induction with IPTG produced cultures that showed detectable expression of KillerRed and mRFP1, however the expression levels were consistently lower for KillerRed despite identical conditions and similar spectral properties. The maturation rate of KillerRed is longer than mRFP1 so it was kept at 4ºC overnight before photobleaching experiments. After sequence analysis, it was determined that the KillerRed is not codon optimized for E. coli and contains 13 rare proline codons, which may cause low translational efficiency and ultimately, low yield of protein expression. Despite this, photobleaching of both proteins were performed in vivo and the viable counts determined. Originally using various controls, it was determined that the lamp's light is non-toxic to the cells and therefore, killing can be attributed to KillerRed expression. As shown in our Results page, we see a 50% decrease in cell viability after 5 hours of photobleaching. This demonstrated that KillerRed is capable of killing bacteria while the negative control, mRFP1, often grows in the presence of light. All of our photobleaching experiments were performed while the cells were log-phase growth, which may differ from previous literature protocols that report large percentages of killing. It is likely that superoxide dismutase is being synthesized in log-phase growth and therefore is able to limit the effect of the ROS.

Figure 1:The plasmid construct we use for expression consists of the <partinfo>BBa_R0010</partinfo> lac promoter, the <partinfo>BBa_B0034</partinfo> 100% efficiency RBS standard and either the KillerRed BBa_K1184000 coding sequence or mRFP1 <partinfo>BBa_E1010</partinfo> coding sequence.

Future Directions

This project laid down the framework for producing temperate phages capable of killing bacteria, thereby circumventing the issue of lysogeny for phage therapies. However, KillerRed as it exists in the Parts Registry is not codon optimized for bacteria and therefore may difficult to get robust expression off of a single copy gene (in the case of a lysogen). We have been able to demonstrate expression of mRFP1 in the prophage but due to the nature of the KillerRed construct, similar expression levels were not obtained. Stronger promoters and ribosome binding sites can be utilized for expression of a codon-optimized KillerRed gene in a prophage to test for killing and therefore, potential phage therapeutic applications. Different temperate phages can be engineered as well to target different organisms so that a cocktail of phages can be produced to kill pathogenic bacteria.