Composite

Part:BBa_K5137501

Designed by: Sazzad Shahrear   Group: iGEM24_Aalto-Helsinki   (2024-10-01)


Drug Component (alternatively names as pYY3c-L-CfaN)

General Biology

This composite protein is the component for drug delivery of our advanced targeted therapy. SpyCatcher is a protein fragment designed to covalently bind with SpyTag, facilitating highly specific and stable interactions with a target component. This binding mechanism allows for precise control over the attachment of therapeutic elements. ADF3 contributes to liquid-liquid phase separation (LLPS) when incubated with our Target component, aiding in the aggregation of molecules, which can improve the efficacy of drug delivery within cells[1]. Additionally, CfaN plays a key role in the system by providing a functional site for ligation with drug payloads[2], enabling the delivery of therapeutic agents directly to the target. Together, these protein fragments create a versatile and dynamic system for targeted therapy, improving the efficiency and specificity of drug delivery.

Cloning Stategy

Overview

We utilized the Golden Gate assembly method to construct this composite part. This method allows for the directional and simultaneous assembly of multiple DNA fragments in a single reaction. By designing compatible overhangs at the ends of each fragment, we ensured the correct assembly of our novel fragment (Spycatch with CfaN at C-terminal) and existing one (Spycatch with ADF3) (Figure 1). The efficiency and accuracy of Golden Gate assembly made it an ideal choice for assembling our complex multi-part design.

Target component
Figure 1: Schematic diagram of Target component

Amplification of Inserts

To amplify the individual inserts for our composite part, we employed the PCR method. All of our gene fragments, including the SpyCatch 002, ADF3, and CfaN, was amplified using custom-designed primers. We also amplified our backbone to insert the overhang. These primers were specifically designed to include desired overhangs which prevent self ligation between insert. At the same time, these overhangs ensured that each fragment would be inserted into the vector in the correct orientation and position during assembly. By optimizing the PCR conditions and verifying the product sizes, we successfully generated the necessary inserts with precise overhangs, enabling seamless integration of the components into the final construct (Figure 2).

Primer sequence for amplifying SpyCatch 002, ADF3, and CfaN 
SpyCatch - L - CfaN F:  AGG TCT CAA TGG TTA CCA CAC TGA GCG GTC TGA GTG GTG AAC AGG GTC CGA 
SpyCatch - L - CfaN R: AGG TCT CAC GAG TCA AGG CAG ACC GTC GAC CTG TTT GAG GTC CAG ACC ACG TTC  

Primer sequence for amplifying pYY3C as backbone for Drug component construction
pYY3C - Fragment 1 – F: AGG TCT CAT ATG CAC CAT CAC CAT CAT CAC GGT GCA ATG GTT ACC ACA CTG AGC GGT CTG 
pYY3C - Fragment 1 – R: AGG TCT CAC CAT TGC GGA GCT AGA ATT CGC AAC GGT GCT TGC TGC ACT C
Target component
Figure 2: Workflow of construction of Drug component

Golden Gate Assembly

Following amplification, the insert and backbone were combined in a single reaction. The Golden Gate assembly relies on type IIS restriction enzymes that cut outside of their recognition sites, allowing for precise ligation of inserts into the backbone. In our experiment, we used Thermo Scientific™ FastDigest Eco31I for restriction digestion and Invitrogen™ T4 DNA Ligase for ligation. All reaction reagent and DNA were mixed by pipetting and incubated at 37°C for 2 hours.

Bacterial transformation

The assembled construct was then transformed into E. coli NEB Stable Competent Cell via heat shock method. The cells were thawed on ice and homogenized by tapping before adding the DNA. 1 μL of plasmid DNA was added to the cell suspension and carefully mixed by flicking the tube 4-5 times and incubated on ice for 30 minutes. Heat shock at 42°C was introduced for 45 seconds and the cells were placed on ice for 5 minutes. The bacteria were recovered by adding 950 μL NEB 10-beta/Stable Outgrowth Medium into the mixture and placed at 37°C shaker incubator with rigorous shaking (250 rpm) for 1 hour. The bacteria were spread on antibiotic plate and incubated at 37°C for overnight.

Verification of the insert sequence

We employed colony PCR and whole plasmid sequencing to confirm successful integration of the insert into the plasmid backbone after successful bacterial transformation. We sequenced our plasmid on Illumina platform. The plasmid sequence was assembled from fastq file. The assembled sequence was then aligned with reference plasmid sequence and identity was 100%.

Expression and validation of the protein

Expression of the protein

The Drug component protein was expressed in E. coli BL21. The gene encoding the protein was cloned under the control of T7 promoter. The bacteria were cultured in LB broth supplemented with Kanamycin (50 µg/ml). The bacteria were cultred at 37°C for 4 hour without any inducer. After the OD600 reached 0.6, IPTG was added and incubated for 20 more hours.

Testing LB and TB as production culture media

The crude cell lysate from each media was analyzed by running the sample in SDS-PAGE. For better resolution, 10% resolving gel was used. The gel was run at 200 V and 400 mA for 60 min. For better resolution, 10% resolving gel was used. The gel was run at 200 V and 400 mA for 60 min. The gel was stained with Coomassie blue and then destained twice to reduce blue background. The on-target band is not too visible in this screening (Figure 3).

Target component
Figure 3: Protein bands on SDS-PAGE from 2 production media and OD600 measurement

FPLC Protein Purification and Concentrator

Despite the setback, we proceeded with mass protein production. Since eGFP plasmids are being expressed more in TB media, we continued with large-scale protein production in 500 mL TB media and used the pellet for lysis and protein purification. Following FPLC with ÄKTA Go, the fractions from wash and elution steps were screened with SDS PAGE gel. The results are shown on Figure 4.The on-target band appears to be faint and proceeded with concentrator process (Pierce Protein Concentrator PES, 50 kDa cut-off.)

Target component
Figure 4: Fractions screening for pCfaC-CGG8 FPLC result

LLPS formation through fluorescence microscope

Functionality test for this drug component is being done in an identical way with pYY8aTv1. Required components to form LLPS were combined and mixed in 0.2 mL PCR tube with the order: 

- 2 uL Doxorubicin-conjugated CfaC-CGG8 
- 2 uL modified Adf3 as drug component 
- 4 uL modified Mfp1 as target component
Small aliquot (2 uL) was pipetted to 0.17 mm thin rectangular cover glass (25.4 mm × 76.2 mm) as object slide and fixed by pressing round-shaped cover glass on it. Following that, the slide was mounted and visualized by Axiovert 5 fluorescence microscope with GFP wavelength (±480 nm excitation wavelength) for fluorescent picture. Bright field image was also captured on the same location for discriminating doxorubicin-negative and positive LLPS. Fluorescence LLPS is observed and we concluded that the site-specific conjugation was happening despite the other drug payload partner (CfaC-CGG8) is forming multimeric structure.

Target component
Figure 7: Fluorescence signal from LLPS with target and Dox-conjugated drug components. Negative control is shown at the bottom row.

References

  1. Yin, Y., Roas‐Escalona, N. and Linder, M.B. (2024) ‘Molecular engineering of a spider silk and mussel foot hybrid protein gives a strong and tough biomimetic adhesive’, Advanced Materials Interfaces, 11(8). doi:10.1002/admi.202300934.
  2. Stevens, A.J. et al. (2016) ‘Design of a split intein with exceptional protein splicing activity’, Journal of the American Chemical Society, 138(7), pp. 2162–2165. doi:10.1021/jacs.5b13528.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 419
    Illegal PstI site found at 635
    Illegal PstI site found at 827
    Illegal PstI site found at 1037
    Illegal PstI site found at 1238
    Illegal PstI site found at 1376
    Illegal PstI site found at 1589
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 419
    Illegal PstI site found at 635
    Illegal PstI site found at 827
    Illegal PstI site found at 1037
    Illegal PstI site found at 1238
    Illegal PstI site found at 1376
    Illegal PstI site found at 1589
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 419
    Illegal PstI site found at 635
    Illegal PstI site found at 827
    Illegal PstI site found at 1037
    Illegal PstI site found at 1238
    Illegal PstI site found at 1376
    Illegal PstI site found at 1589
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 419
    Illegal PstI site found at 635
    Illegal PstI site found at 827
    Illegal PstI site found at 1037
    Illegal PstI site found at 1238
    Illegal PstI site found at 1376
    Illegal PstI site found at 1589
    Illegal AgeI site found at 346
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
Parameters
None