Overview

1.We designed 14 basic parts and 3 composite parts in total. Among these parts, our favorite basic part is BBa K4421003 (scfv library with 10 individual colonies) and our favorite composite part is BBa K4421027 (CAR_library).

2.We added new documents on the existing part BBa K4040034 ,contributing to future teams.

3.We also improved part BBa K3244013 and BBa K3755010, adding a new improved part BBa K4421031.

Basic parts

Our basic part collection is mainly composed of two groups: the components of CAR and the components of our genetic circuit, namely, the kill-switch circuit. The former group consists of the scFv library which is our favorite and 10 scFvs: cetuximab, trastuzumab, CH65, 9.8B, 2F5, 7D11, 8D6, omalizumab, TE33, and R10. The latter group consists of iCASP9, 2A peptide and NFAT-RE promoter.

composite parts

Our composite part collection includes CAR_library, NFAT_RE-Gal4-KRAB and pSV40-UAS-iCASP9-2A-GFP.

Favorite parts

Among these parts, we like BBa K4421003 (scfv library with 10 individual colonies) most, which provides novel ideas for whoever interested in CAR and tumor heterogeneity.

Scfv library with 10 individual colonies demonstrates the concept of antibody library. As we all know, developing and evolving tumors contain diverse and substantial antigens, which form an antigen library, rather than a single kind of antigen, embodying the characteristic of tumor heterogeneity. So the traditional ideas to depend on specific CARs are unreliable and inappropriate. In our project, we use an antibody library to combat with the antigen library extensively, addressing tumor heterogeneity. Future teams or researchers who are devoted to tumor heterogeneity and CAR, can borrow from our part BBa_K4421003 and idea of antibody library.

Our favorite composite part is BBa K4421027(CAR_library), which consists of BBa_K4421003 and other CAR elements.

part contribution

When searching for project-related part information in the registry, we found in the part BBa K4040034, we can add new information about its usage.

Here shows what we added:

Usage supplementation: HER2, also known as ErbB2, is one of four members (HER1–4) of the epidermal growth factor receptor or HER family. All HER receptors share a similar structure: an extracellular ligand-binding domain, a short hydrophobic transmembrane region, and a cytoplasmic tyrosine kinase domain. Hetero- or homodimerization of HER receptors, induced by ligand binding or receptor overexpression, leads to the activation of the receptor kinase and to the subsequent phosphorylation of several tyrosine residues. In turn, these phosphorylated tyrosine residues, located within the carboxyl terminus of the receptors, recruit mediators and activate signaling pathways that result in the modification of the cell growth, differentiation, and survival.

When referencing as truncated receptor, it is usually means the p95HER2, which has kinase activity in the absence of the trastuzumab-binding extracellular domain led us to hypothesize that p95HER2–expressing tumors may be resistant to trastuzumab but sensitive to the inhibitory effects of lapatinib, a low–molecular-weight tyrosine kinase inhibitor (TKI) of HER2 that has activity in patients with HER2–expressing tumors that are resistant to trastuzumab.

However, this part includes the Truncated HER2 without the ICD domain, which can be used as a marker for engineered synthetic immune cells or other cells, such as CAR-T cells or liver cells. When used as a marker, the engineered cell can be easily cleared with anti-HER2 antibodies, such as Trastuzumab,or other HER2 targeting methods.

Part improvement

In our project, we designed a kill-switch circuit which involved NFAT response element. So we searched in the registry for existing NFAT response element part. BBa K3244013 designed by iGEM19_AFCM-Egypt and BBa K3755010 designed by iGEM21_ShanghaiTech_China were available for us to improve.

In Part: BBa_K3244013, they generally introduced the biological background of NFAT response element, but with no experiment results.

In Part: BBa_K3755010, they cotransfected NFAT-RE+minP+EGFP(Part:BBa_K3755014) and Piezo1.1(Part:BBa_K3755006,calcium signal provider) into HEK 293 cells. Through fluorescence colocalization, they observed some cells had produced EGFP successfully, which meant NFAT and NFAT response element functioned well.

We performed 33 random mutations on BBa_K3755010.

Taking BBa_K3755010 as control, results showed the response strength of mutation 13 was significantly higher than the control and other mutants, promising to achieve ideal results in our experiment.

>BBa_K3755010

ggaggaaaaa ctgtttcata cagaaggcgt ggaggaaaaa ctgtttcata cagaaggcgt ggaggaaaaa ctgtttcata cagaaggcgt

>mutation 13 (26: g→c)

ggaggaaaaa ctgtttcata cagaacgcgt ggaggaaaaa ctgtttcata cagaaggcgt ggaggaaaaa ctgtttcata cagaaggcgt