In the CTC-FAST project, we aim to develop the parts for CTC-capture and CTC-labeling.
To generate the tetrahedral single-stranded DNA (ssDNA) for the CTC-capture, we re-construct the rolling cycle replication (RCR) in E. Coli. The parts associated with RCR include the RCR initiation protein RepA (BBa_K4674002), RCR start motif (BBa_K4674007), RCR stop motif (BBa_K4674008) and ssDNA binding protein (SSBP; BBa_K4674003).
To make the CTC labeling more sensitive, we used the mGreenLarten (mGL) protein (BBa_K4674001), which has higher brightness than canonical eGFP, to label the captured CTCs.
Together, we believed that the RCR associated parts and the part of fluorescence protein with stronger brightness would contribute to the iGEM teams in the future.
| Part number | Name | Description | Length |
|---|---|---|---|
| BBa_R0010 | Lac promoter & operator | The lac promoter operator sequence is utilized in the control of gene transcription. | 200bp |
| BBa_K3778018 | T7 terminator | The T7 terminator is a sequence from bacteriophage T7 which allows efficient transcription termination. The T7 promoter and terminator are commonly used to regulate gene expression of recombinant proteins. | 39bp |
| BBa_K3778013 | RBS | The ribosome binding site (RBS) sequence serves as a rendezvous point for the ribosome, which moves alone the mRNA and proceeds to translate mRNA from the following start codon. | 23bp |
| BBa K4674004 | PBSII-Zif268 fusion protein | Zif268 is a naturally occurring 3-finger ZF that has been extensively studied structurally and biochemically. It binds the 9 bp sequence 5'-GCG TGG GCG-3'. PBSII is designed 3-finger ZFs assembled from predefined modified ZF domains, and recognize the sequences 5'-GTG TGG AAA-3' | 558bp |
| BBa_K4674002 | RepA | The RepA is a replication initiator protein that initiate the process of rolling circle replication (RCR) of the R-plasmid, pC194. RepA binds to the double-stranded origin (DSO) and creats nick on one of the DNA strand. The DNA polymerase use unnicked strand as template to elongate the nicked strand. Finally, the elongated nicked strand is completely replaced by the newly synthesised strand, and the replication initiator enzyme will then ligate the elongated nicked strand into a circular DNA. | 690bp |
| BBa_K4674003 | Single-strand binding protein (SSBP) | SSBP forms honotetraner and exhibits high affinity towards single stranded DNA (ssDNA). SSBP also prevents the formation of inhibitory secondary structures in the exposed lagging-strand of DNA during replication. | 366bp |
| BBa_K4674000 | mGreeen Lantern (mGL) | mGreenLantern is a type of green fluorescent protein with excitation and emission peaks at 503 and 514 nm, respectively. It differs from EGFP by 21 mutations. It possesses a higher cellular brightness (630% higher than EGFP) and matures rapidly (207% faster than EGFP). | 720bp |
| BBa_K4674001 | mGL-4A-C7 | This part is modified from the BBa_K4674000 (mGL). The C-terminal of BBaK467000 (mGL) is fused with a linker composed of 4 alanines, following a C7 peptide for folate receptor alpha recognization. | 768bp |
| BBa_K4674005 | C7 dodecapeptide | The sequence of C7 peptide is Met-His-Thr-Ala-Pro-Gly-Trp-Gly-Tyr-Arg-Leu-Ser, which is developed from the phage library screening. The ability of C7 peptide to recognize FRα is proved by FACS analysis of SKOV3 cell lines in vitro and the in vivo tumor staining. The equilibrium dissociation constant (KD) value between C7 and FRα was 0.3 μM. | 36bp |
| BBa_K4674007 | RCORI-105 | The motif RCORI105 is 105 bp sequence at the 3' terminus of double-stranded origin of R-plasmid pC194. RCORI-105 serves as the start point of RepA medated circular ssDNA synthesis in rolling circle replication. | 105bp |
| BBa_K4674008 | RCORI-65 | The motif RCORI-65 is 65 bp sequence at the 5' terminus of double-stranded origin of R-plasmid pC194. RCORI-65 serves as the stop point of RepA medated circular ssDNA synthesis in rolling circle replication. | 65bp |
| BBa_K4674006 | Tetrahedral ssDNA with cis-auto splicing motif | The tetrahedral ssDNA is designed to self-assemble into a DNA tetrahedon in a complemetary mannrer. The 3' terminus is following by cis-autosplicing sequence. | 330bp |
| Part number | Name | Description | Length |
|---|---|---|---|
| BBa_K4674009 | The eGFP-C7 expression cassette | This expression cassette is regulated by lac operator. The N-terminal of eGFP-4A-C7 protein is fused with a 6xHis-tag and a thrombin cleavage site. | 1027bp |
| BBa_K4674010 | The mGL-C7 expression cassette | This expression cassette is regulated by lac operator. The N-terminal of mGL-4A-C7 protein is fused with a 6xHis-tag and a thrombin cleavage site. | 1027bp |
| BBa_K4674013 | The PBSII-Zif268 expression cassette | The expression cassette is composed of T7 promoter, Lac operon, RBS, PBSII, Zif268, and T7 terminator. | 822bp |
| BBa_K4674011 | The RepA and SSBP expression cassette | The expression cassette is composed of T7 promoter, Lac operon, RBS, His tag, SBP, RBS, RepA, and T7 terminator. | 1341bp |
| BBa_K4674012 | Tetrahedral ssDNA - RCR version | The version is composed of RCORI-65, Cis-autosplicing sequence, Tetrahedral ssDNA, and RCORI-105. | 500bp |
Komura, R., et al. (2018). High-throughput evaluation of T7 promoter variants using biased randomization and DNA barcoding. PloS one, 13(5), e0196905.
https://doi.org/10.1371/journal.pone.0196905
Jia, Y., et al. (2021, April 8). DNA-Catalyzed Efficient Production of Single-Stranded DNA Nanostructures. ScienceDirect.
https://doi.org/10.1016/j.chempr.2020.12.001.
Jia, Y., et al. (2021, April 8). DNA-Catalyzed Efficient Production of Single-Stranded DNA Nanostructures. ScienceDirect.
https://doi.org/10.1016/j.chempr.2020.12.001.