Our results
To address the problem of LPS induced septic shocks, we designed a phage which should be able to decrease the LPS release when killing a bacteria. On the way of the phage becoming reality, many exciting achievements have been made which are shown below. This includes our characterization part for the registry, the preparation for the homologous recombination, the characterization of the wild type phage and our modeling result.
Characterization
To help characterize the iGEM registry, the three constitutive promoters BBa_J23101, BBa_J23105 and BBa_J23109 were compared in the cell free expression system myTXTL® Sigma 70 Master Mix Kit.
The measured fluorescence is presented in fluorescence units [FU].
| Time [h] |
101 [FU] |
105 [FU] |
109 [FU] |
| 1,3 |
8 |
6,5 |
6 |
| 2,8 |
7,5 |
6,5 |
6 |
| 4,8 |
28 |
6,5 |
6 |
| 8,0 |
57 |
14 |
7 |
Table 1. Fluorescence of mCherry at assay 1
The data clearly shows that the respective expression strength in E. coli (101>105>109) remains the same in this cell free expression system.1 However, the difference between 101 compared to 105 and 109 is far more pronounced than the difference between 105 and 109. To see the differences better, especially between 105 and 109, a second experiment was conducted. For this, plasmid DNA with higher concentration was produced. Through this, a higher concentration of cell free reaction mix could be used as well.
| Time [h] |
101 [FU] |
105 [FU] |
109 [FU] |
| 3,45 |
534 |
7,5 |
6 |
| 5,03 |
937 |
9,5 |
5,5 |
| 7,45 |
1310,5 |
18,5 |
4,5 |
| 9,45 |
1082 |
19 |
5,5 |
Table 2. Fluorescence of mCherry at assay 2
Interestingly, a drop-off in fluorescence past 7.5 hours can be seen for 101. This might be the result of experimental errors and different operators. This seems as a more coherent explanation than a decay of protein. Even though the incubation time was longer and the concentration of the cell free expression system higher, the difference between 105 and 109 is hardly visible. In fact, 109 showed no activity at all as can be seen in table 2.
Conclusion
To produce a notable amount of a protein of interest, the promoter 101 seems to be the best choice. 105 shows some activity compared to 109 which produced no measurable amount of protein. Just like when used in bacterial hosts in previous characterization experiments, BBa_J23101 showed higher expression strength than BBa_J23105 and BBa_J23109.2 This result seems valid. However, this result is meaningful for this very expression system.
Preparation for homologous recombination
Assembling of the Lambda RED Plasmid
The major part of our work in the wet lab was the assembly of a plasmid containing the Lambda RED system. Due to high toxicity of the expressed proteins, the tight regulation of recombination plasmids is important.3 It would have been possible to use the tight promoter pBAD.4 However, since the used E.coli strain Bl21(DE3) carries the T7 polymerase under control of the lac-operon it was decided to control the plasmid as well with a lac-operon. Even though it is not quite the promoter we used, such a promoter can be found in the iGEM registry already with the part number BBa_K2406020. More details to the parts we used can be found here. This results in two expression barriers as well as a permanent concentration of lac repressor in the cell.5
The plasmid was assembled in three cloning steps. First, the proteins exo (BBa_K3514001), beta (BBa_K3514002) and gam (BBa_K3514003) were cloned into a backbone to add desired cloning sites. These proteins are part of our contribution to the registry. Second, a promoter and a terminator were added to each gene. This resulted in three open reading frames. Thirdly, these three frames were assembled in a single plasmid with a kanamycin resistance gene (BBa_K3514004).
Figure 3: recombination plasmid map BBa_K3514004
Figure 4: Lane 5 shows the with PstI digested recombination plasmid
Production Strain
The designed phage lacks its Major Capsid Protein 10 (see also Project description). Therefore, the phage can be cultivated on a strain which supplements the Major Capsid Protein 10. Hence, an open reading frame including a lac-promoter was designed, assembled via golden gate cloning and transformed in E. coli BL21(DE3) which produces the Major Capsid Protein 10.
Production of recombineering substrate
To use the Lambda RED system for homologous recombination, a substrate is needed. Therefore, the Protein Human Plasma Gelsolin, a synthetic antimicrobial peptide and the binding site of Human Plasma Gelsolin to LPS (AA 160-169) were amplified via PCR. After that, the products were desalted for electroporation with “Monarch® PCR & DNA Clean-up Kit “.
Figure 5: Lane 2-Human Plasma Gelsolin, Lane 3-synthetic Peptide BBa_K3514000, Lane 4-gelsolin binding site
The other substrate for engineering the T7 Phage is of course its own DNA. Therefore, the Phage was cultivated, purified via a PEG-precipitation and the DNA extracted via a Phenol-Chloroform extraction.6 To prove the functionality of the phage DNA, it was electroporated in E. coli Bl21 DE3. Unfortunately, no consistent plaque growth was monitored.
Characterization of the wild type T7 phage
Since the engineered phage would lack the gene for its major capsid protein, its growth behavior is likely to change. To be able to monitor the altered phage compared to the wild type, we collaborated with PHOCUS, the iGEM team of the TU Delft, who had similar concerns. Each team developed a growth assay which was shared with the other team. Unfortunately, both teams had no isolated engineered phages which could be used for experiments. Therefore only the wildtype was used.
Team TU Delft developed a plaque size assay. The growth of plaques made by phages is monitored over time by measuring the diameter.
Figure 6: Plaque size monitored by Team Vienna
Figure 7: Plaque growth monitored by Team Delft
The results showed a growth rate of 0.37 mm/h for Team TU Delft and 0.48 mm/h for Team BOKU-Vienna, respectively. Since both teams used the same phage, the result shows a lack of robustness and validity. This was expected since both teams are not experienced with the development of protocols and the experiments were only conducted once. However, more data would be needed for further protocol development.
The infection dose assay should determine the rough amount of phages which are needed to clear a growing batch process of the susceptible organism. This result can be useful for later upscaling in process development when larger amounts of the phage are needed. As an optimal inoculation dose, we determined a ratio of 1.3 PFU/bacteria to produce phages in 30 minutes. Team TU Delft monitored only a slight decrease in bacterial growth but no clearance. Further experiments and repetitions are needed to find the reason for this result. The decrease in bacteria concentration after phage inoculation can be seen in figure 8.
Figure 8: The decrease in bacteria concentration after different phage doses in 4 ml bacteria
Modeling
To conduct the characterization experiments and to produce phage DNA for recombination, phages are needed. These are produced in bacteria batch cultures. To follow the question of how to produce as many phages as possible, a simple modeling experiment was performed. (more details can be found on our Modelling site)
Figure 9: Phage growth at different inoculation timepoints
The model concluded that the highest phage concentration can be achieved when introducing the phages to the bacterial culture 40 – 50 minutes after inoculation.
1http://parts.igem.org/wiki/index.php?title=Part:BBa_J23101 (last seen on 26.10.2020)
2John Anderson Group: iGEM2006_Berkeley. ‘Part:BBa_J23101’, n.d..
3,4Sharan, Shyam K, Lynn C Thomason, Sergey G Kuznetsov, and Donald L Court. ‘Recombineering: A Homologous Recombination-Based Method of Genetic Engineering’. Nature Protocols 4, no. 2 (February 2009): 206–23. doi: 10.1038/nprot.2008.227.
5Dubendorf, John W., and F.William Studier. ‘Controlling Basal Expression in an Inducible T7 Expression System by Blocking the Target T7 Promoter with Lac Repressor’. Journal of Molecular Biology 219, no. 1 (May 1991): 45–59. doi: 10.1016/0022-2836(91)90856-2.
6Sayers, J. R., and F. Eckstein. ‘Properties of Overexpressed Phage T5 D15 Exonuclease. Similarities with Escherichia Coli DNA Polymerase I 5’-3’ Exonuclease’. The Journal of Biological Chemistry 265, no. 30 (25 October 1990): 18311–17.
