Composite

Part:BBa_M36459:Design

Designed by: Ian Lewis, Max Whitmeyer   Group: Stanford BIOE44 - S11   (2013-12-17)


PoPS -> Cell Lysis


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Basic plasmid design

The sequence was altered in a few places in order to avoid repeats and palindromes. Also, the sequence had to be edited further when the Ribosome Binding Sites(RBS) were added to avoid repeats. It's also important to note that the part utilizes one Bicistronic Design(BCD) and two Monocistronic Designs(MCD) for the RBS's. Three BCDs would have been used but all BCDs have significant overlapping sequences and only one could be used to avoid repeats. All RBS's were chosen for low to medium expression level and low variance.

To accomplish the goal of induced cell lysis, various cell lysis DNA sequences were examined on the BioBricks part registry. One particularly intriguing DNA sequence included three encoded proteins, an S105 protein (Holin), an R protein (Endolysin), and an Rz protein. These three proteins combine to cause total cell lysis. This sequence was edited to avoid palindromes and repeating sequences greater than or equal to 10 base pairs.

Holins consist of an extremely diverse group of proteins that all lead to cell lysis. Holins are unique in that they are “time-specific”; they accumulate over time and then lyse the cell membrane all at once (Grundling). The endolysin kills the cell by disruption of the cell wall (Payne). This is important to include in our sequence since E. Coli has a cell wall. The Rz protein helps the holin perforate the cell membrane to cause lysis. All three of these proteins are necessary for complete cell lysis of E. Coli (Berry).

A Bicistronic Design (BCD) was used for the first Ribosome Binding Site (RBS) in order to accurately predict transcription levels. This is especially important when dealing with cell induced death as an unexpected high level of transcription could lead to premature cell death before enough E. Coli was produced to do the testing. Thus, a BCD with a relatively low strength and extremely low variance was chosen for the DNA construct. The Endolysin and Rz protein were both paired with a Monocistronic Design (MCD) as there is too much similarity in the sequences of the BCDs to synthesize multiple in the same construct. However, the MCDs chosen had low expression and low variance as well. The MCDs and BCD were found using the supplementary data table to the paper “Precise and reliable gene expression via standard transcription and translation initiation elements”. For the BCD associated with the holin protein, we chose pFAB1692, with a relative strength of ~75 and standard deviation of ~3. For the MCD prior to the Endolysin, we chose apFAB706, with a relative strength of ~66 and a sd of ~2. And finally, for the MCD placed before the Rz protein, we chose apFAB720, with a relative strength of ~66 and a sd of ~6.

The terminator used for the cell lysis DNA construct was classified and rated in the journal article "Measurement and Modeling of Intrinsic Transcription Terminators”. It was the second strongest terminator that was tested with ~99% termination efficiency. Since this terminator is being placed at the end of our 3 protein sequence, there is no reason to use a weak terminator.

Source

The sequences for all three proteins were found from Part BBa_K124017 in the BioBricks registry. All Ribosome Binding Sites and the Terminator found from the biofab website in the data section.

References

Grundling, Angelica, Michael D. Manson, and Ry Young. "Holins Kill Without Warning."PNAS (2001)

Payne KM, Hatfull GF (2012) Mycobacteriophage Endolysins: Diverse and Modular Enzymes with Multiple Catalytic Activities. PLoS ONE

Berry, Joel Dallas. The Final Step in Phage Lysis: The Role of the RZ-RZ1 Spanin Complex in the Disruption of the Outer Membrane. Texas A&M. (2010)