Contribution page!

Overview

In CTC-FAST project, we contribute to the iGEM community by adding some new parts for expressing the stronger green fluorescence protein, mGL, and some unusual parts to express and purify single-stranded DNA (ssDNA).

We also build up the hardware for automatic washing and incubation, the CTC-FAST device. In CTC-FAST device, we apply the 3D CAD software, the Solidworker, to generate several components, including the main chamber, the trident microchannel, the microchannel box and the tri-syringe holder. We left the blueprint for the iGEM teams in the future.

The new basic part

  • mGreenLantern (mGL) fluorescent protein (BBa_K4674000):the new protein possesses a higher cellular brightness (630% higher than eGFP) and matures rapidly (207% faster than eGFP).

    • ▲ The spectrum of mGL excitation and emission is quated from fpbase (Lambert, 2019)


  • mGL-4A-C7 fusion protein (BBa_K4674001): the mGL protein for labeling folate receptor ɑ (FRɑ) positive circulating tumor cells (CTCs).

Compared to the conventional eGFP in laboratory application, mGL possesses advantages in terms of brightness, and its excitation and emission peak values are compatible with common eGFP filters. This theoretically makes mGL a highly suitable alternative to eGFP for fluorescence labeling in related experiments.

Using these basic part, we generated the composite part (BBa_K4674010) and confirmed that the mGL fusion protein expression could be induced through the traditional pET system. Furthermore, we successfully purify the mGL fusion protein by FPLC. The stability of mGL fusion protein is compatible to the conventional eGFP, and the strength of fluorescence is stronger than eGFP. The mGL fusion protein could be monitored under microscope by the standard filter for eGFP. After confirming the authenticity of its properties and its experimental feasibility, we are more confident that mGL has the potential to become a more widely utilized fluorescent labeling material in the future iGEM community, making related experiments more smoothly, efficiently and clearly

The new parts for ssDNA expression in vivo

  • The RepA protein (BBa_K4674002): The RepA is a replication initiator protein that initiates the process of rolling circle replication (RCR) of the R-plasmid, pC194. The RepA protein could recognize RCORI-105 and RCORI-65 as start and stop motifs, respectively, and replicate the intervaling sequence into ssDNA in bacterium.
  • The Single-strand binding protein (SSBP; BBa_K4674003): SSBP forms honotetraner and exhibits high affinity towards ssDNA. SSBP also prevents the formation of inhibitory secondary structures in the exposed lagging-strand of DNA during replication.
  • RCORI-105 (BBa_K4674007): The motif RCORI-105 is 105 bp sequence at the 3' terminu of double-stranded origin of R-plasmid pC194. RCORI-105 serves as the start point of RepA mediated circular ssDNA synthesis in RCR.
  • RCORI-65 (BBa_K4674008): The motif RCORI-65 is 65 bp sequence at the 5' terminu of double-stranded origin of R-plasmid pC194. RCORI-65 serves as the stop point of RepA mediated circular ssDNA synthesis in RCR.

In the iGEM community, these are the first parts associated with ssDNA production. We generated two composite parts: The composite part BBa_K4674011 is for the inducible SSBP and RepA protein expression. The composite part BBa_K4674012 is the ssDNA cassette for RepA recognition.

The composite part of RCR expression cassette

▲ The composite part of RCR expression cassette


After the IPTG induction, the RepA protein are induced to initiate the ssDNA from RCORI-105 to RCORI-65. The induced SSBP protein could protect the ssNA from degradation, and the his-tag on the SSBP protein could be applied to purify SSBP protein as well as the binding ssDNAs.

The functions of composite part BBa_K4674011 and BBa_K4674012

▲ The functions of composite part BBa_K4674011 and BBa_K4674012


The blueprints of hardware components

All works could be downloaded here(all works are generated by Soliworkers): CCU-iGEM-Contribution

  • Main Chamber: The main chamber is in a diamond shape to avoid the accumulation of flowthrough in the chamber. The total volume is 39 ml. A circle with a diameter of 10 mm was opened in the center of the upper surface as a hole for injecting blood samples, and holes with a diameter of 5 mm were drilled on both sides of the chamber wall for liquid circulation.

    • ▲ The design of the main chamber


  • The trident microchannel: To make the faying surface between syringe and microchannel tight, we make the trident microchannel with the main flow channel at 150 microns and the other two channels at 300 microns.

    • ▲ The design of the trident microchannel


  • Microchannel box: The purpose of a microchannel box is to hold the microchannel and keep the microchannel in the same plane as the liquid propulsion device.

    • ▲ The design of the microchannel box


  • Tri-syringe holder: The purpose of tri-syringe holder is to keep the steady and synchronous injection of three syringes when the step motor propels the plug.

    • ▲ The design of the Tri-syringe holder