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Team:Montpellier/Parts

Team:Montpellier - 2020.igem.org

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Team:Montpellier

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PARTS



Because of the COVID-19 pandemics, our wetlab period was reduced, and the ordering procedures were slowed down. Thus, we managed to build our synthetic phage from two parts that were already available in the lab: the plasmid “pFab217” that contains sfGFP and kanamycin resistance and the phagemid p7560 which is usually used for DNA origami applications but still contains the phage M13 genes. We chose this phage because it doesn’t lyse the bacteria. We used the phagemid (p7560) as a vector and added the kanamycin antibiotic resistance from the insert (pFab217). We opted for a Gibson Assembly cloning strategy. So we designed and ordered two pairs of primers with complementary ends to selectively amplify and join (1) the M13 genes and (2) the kanamycin resistance and GFP genes. We then proceeded with the Gibson Assembly to build the phagemid we wanted to use for our proof of concept.

For the next year, we plan to build our fully functional vector, once we have tested the GFP-expressing phage for our proof of concept experiments. We aim to replace sfGFP with various genes of therapeutic interest. We decided to use an inducible pTac promoter, and terminators B0014 and J61048 to stop all undesired transcription coming from outside our cassette.

The genes of therapeutic interest that we chose are DacA, Azurin, and a VHH targeting PD-L1.

DacA is the protein that we chose first for this project, this gene will help the bacteria to activate the STING pathway by producing di-AMPc [1].

Azurin is a gene that produces a peptide with antitumor activity when expressed by tumor-targeting bacteria (REF). The studies of standard biological parts of BBa_K2500001 said that “In vitro studies have demonstrated that the cytotoxicity of azurin is due to its ability to raise intracellular levels of p53 by binding and stabilizing it upon internalization” [2]. Azurin is potentially a highly versatile and promising anti-cancer agent as additional mechanisms keep being discovered such as inhibition of angiogenesis and cell invasion [3]. Finally, we will use a VHH to block the PD-L1 from the tumor cells. These ligands help the tumor cells to escape the immune response. This mechanism is called immune checkpoint blockade. There are molecules that either promote or inhibit the action of the T-cells. There exist different types of inhibitory molecules, cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-L1). The VHH will interfere with the immune response blockade by PD-L1.

In order to make those constructs, some basic parts where needed:



Table 1: Basic Parts
Number of the part Type Description Length (bp)
BBa_B0014 Terminator Double terminator B00014 in order to regulate transcription 95
BBa_J61048 Terminator Terminator J61048 in order to regulate transcription 113
BBa_K864400 Promoter Inducible pTAC promoter 36
BBa_J23110 Promoter Constitutive promoter 35
BBa_K3700000 Spacer Spacer in order to clear sequence and make generic primers 40
BBa_K3700001 Spacer Spacer in order to clear sequence and make generic primers 40
BBa_K3700002 Spacer Spacer in order to clear sequence and make generic primers 40
BBa_K3700003 Spacer Spacer in order to clear sequence and make generic primers 40
BBa_J61101 RBS RBS where the ribosome will bind to the RNA for translation 26
BBa_K1223006 Reporter HisTAG in order to detect protein expression by western blot 18
BBa_K105012 Linker Linker in order to link to protein sequences 10
BBa_I746916 Reporter sfGFP coding sequence 711
BBa_K1045003 Enzyme DacA (diadenylate cyclase) coding sequence 411
BBa_K2500001 Protein Azurin coding sequence 384
BBa_K3700005 Secretion signal Secretion signal to export protein out of the cell 60
BBa_K2865001 Antibody VHH PD-L1 nanobody coding sequence 483


Next year we will clone these composite parts into the recombinant phagemid (pFAB) that we made. For the reporter constructs containing GFP, we chose to use super-folder GFP (sfGFP)[4] [5], a robust variant of GFP that folds better than GFP when expressed as fusions with other proteins or poorly folded polypeptides.
For some composites, we need to use a linker, because we want to test 2 different genes in the same composite. The linker that we use is the 10 AA linker K105012 to avoid any steric hindrance between DacA and sfGFP or Azurin and sfGFP.
Below is a list of our prospective composite parts:




Table 2: Composite Parts
Name Design Description
BBa_K3700004 sfGFP linked to the dacA gene with an inducible promoter surrounded by spacers in order to create universal primers.
DacA helps the bacteria to activate the STING pathway by producing di-AMPc5. We will add a GFP to find the bacteria containing correct clones, and to assess protein production easily.
BBa_K3700006 sfGFP linked to the dacA gene with a constitutive promoter surrounded by spacers in order to create universal primers.
DacA helps the bacteria to activate the STING pathway by producing di-AMPc5. We will add a GFP to find the bacteria containing correct clones, and to assess protein production easily.
BBa_K3700007 sfGFP with a secretion signal and an inducible promoter surrounded by spacers in order to create universal primers.
sfGFP with a secretion signal in order to check if the cell can secrete proteins.
BBa_K3700008 sfGFP with a secretion signal and a constitutive promoter surrounded by spacers in order to create universal primers.
sfGFP with a secretion signal in order to check if the cell can secrete proteins.
BBa_K3700009 sfGFP linked to the Azurin gene with a secretion signal and an inducible promoter surrounded by spacers in order to create universal primers.
Verification of the expression and the secretion of the azurin thanks to the sfGFP.
BBa_K3700010 sfGFP linked to the Azurin gene with a secretion signal and a constitutive promoter surrounded by spacers in order to create universal primers.
Verification of the expression and the secretion of the azurin thanks to the sfGFP.
BBa_K3700011 VHH-PDL1 with a secretion signal and an inducible promoter surrounded by spacers in order to create universal primers.
BBa_K3700012 VHH-PD-L1 with a secretion signal and a constitutive promoter surrounded by spacers in order to create universal primers.




References

[1] Leventhal, D.S., Sokolovska, A., Li, N. et al. Immunotherapy with engineered bacteria by targeting the STING pathway for anti-tumor immunity. Nat Commun 11, 2739 (2020). https://doi.org/10.1038/s41467-020-16602-0)
[2] T. Yamada et al. "Bacterial redox protein azurin, tumor suppressor protein p53, and regression of cancer." Proceedings of the National Academy of Sciences (2002): 14098-14103
[3] R. Mehta et al. "A cell penetrating peptide derived from azurin inhibits angiogenesis and tumor growth by inhibiting phosphorylation of VEGFR-2, FAK and Akt." Angiogenesis (2011): 355-369
[4] Raran-Kurussi, Sreejith, et David S. Waugh. 2012. « The Ability to Enhance the Solubility of Its Fusion Partners Is an Intrinsic Property of Maltose-Binding Protein but Their Folding Is Either Spontaneous or Chaperone-Mediated » éd. Bostjan Kobe. PLoS ONE 7(11): e49589.
[5] Overkamp, W. et al. (2013) Benchmarking various green fluorescent protein variants in Bacillus subtilis, Streptococcus pneumoniae, and Lactococcus lactis for live cell imaging. Appl. Environ. Microbiol. 79: 6481-6490 https://www.nature.com/articles/nbt1172