Composite

Part:BBa_K3504010

Designed by: Ahmed Wael   Group: iGEM20_AFCM-Egypt   (2020-10-09)
Revision as of 09:43, 23 October 2020 by Ahmed Wael (Talk | contribs) (→‎Characterization)


Multi-epitope TNBC Vaccine regulated by SGP 30

Part Description

A composite of parts (BBa_K3504004,BBa_K3504007,BBa_K3504020) Which form as a whole a multiepitope vaccine accompanied by Gly-Ala repeats which work in protecting the circuit from immune response attack.

Usage

SG RNA is transcribed from the SG promoter and serves as template for translation of viral structure protein e.g.:capsid,E2 and E1 which gives alpha virus attractive systems for designing self replicating vectors for delivery and expression of heterogeneous genetic information. It’s also used for amplification and replication by the VEEV replication complex SG RNA is transcribed from a promoter located in the alpha virus specific RNA replication intermediate and is not further amplified. SG promoter transcribes RNA encoding proteins of interest and encodes the same (3 CSE ) and poly A tail as the viral genome but contains a different (5 UTR) and lacks the (51-nt CSE). We used it as a control to demonstrates a standard level of RNA synthesis and heterogeneous protein expression. Immune-modulating adjuvants and PADRE (Pan HLA-DR epitopes) sequence were added with epitopes sequence to enhance the immunogenicity. All the epitopes, adjuvants and PADRE sequence were joined by linkers. Heat-shock proteins (hsp) provide a natural link between innate and adaptive immune responses by combining the ideal properties of antigen carriage (chaperoning), targeting and activation of antigen-presenting cells (APC), including dendritic cells (DC). EBNA1 glycine-alanine repeat (GAR) domain when inserted in cis with CTL epitope can inhibit antigen presentation through preventing cytotoxic T-lymphocyte-epitope generation. GAR appears to inhibit the proteosomal degradation of indicator proteins into which the gly-ala repeat is introduced, therefore evades the recognition by CTLs. previous findings show that the Gly-Ala repeat prevents presentation of MHC class I-restricted epitopes through the inhibiting the ubiquitin/proteasome pathway. The miRNA-126 has an already established role in angiogenesis and cancer, but recently in the current issue of Nature immunology, it was reported by agudo and colleagues that this miRNA-126 plays a part in survival and function of plasmocytoid dendritic cells (pDCs).

Characterization


We have made simulations using mathematical modelling techniques which showed expression values relative to those present in literature as shown in figures 1 & 2. These findings suggest that SGP 30 & 15 show the highest expression values with SGP 15 being the highest
We also made simulations using mathematical modelling techniques which showed lower levels of CTLs degredation when adding the glycine-alanine repeats to our circuit compared to its degredation ratio without the repeats.

Figure 1.This figure shows mathmatical modelling simulations illustrations expression variation between SGP30 vs SGP15 which both show high expression however SGP 15 show much higher expression .
Figure 2. shows mvenus expression in different variants of subgenomic promoters.
Figure 3.This Figure shows the sbol format of this composite part.
Figure 5. Using C-IMMSIM simulation models to describe both Humoral and Cellular response of our predicted epitopes in our 1st proposed multi-epitope DNA vaccine for TNBC, Showing Antigen & Immunoglobulins level, B-cell Populations with various isotypes as well as Memory & Not Memory B-Cells and CD4 T-Cell Population & the regulatory T-Cells.
Figure 4. Using C-IMMSIM simulation models to describe both Humoral and Cellular response of our predicted epitopes in our 1st proposed multi-epitope DNA vaccine for TNBC,Showing the CD8 T-Cell Population and NKCs & DCs & MQs & Epithelial Populations.
Figure 6. CTLs degredation ratio between it with the inhibitory effect of Gly-Ala repeat on CTLs attack on the left compared to traditionally without it on the right.











































Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 2300
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 741
    Illegal NgoMIV site found at 765
    Illegal NgoMIV site found at 963
    Illegal NgoMIV site found at 1104
    Illegal NgoMIV site found at 1143
    Illegal NgoMIV site found at 1206
    Illegal NgoMIV site found at 1272
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1634
    Illegal BsaI.rc site found at 2120
    Illegal SapI.rc site found at 1505


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