Difference between revisions of "Part:BBa K2959010"

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As shown in figure 3, bands are presented at the approximate weight of 14.3 kDa which corresponds to AtPFN1 as corroborated by UniProt. Visible bands of AtPFN1 correspond to protein extracts of transformed <i>E. coli</i> BL21 (DE3) induced with different concentrations of IPTG. No band can be appreciated in the negative control column, following the protein ladder, which belongs to a control of untransformed cells, afirming that the bands are indeed AtPFN1. No basal level expression was observed on the uninduced control (transformed cells with no IPTG, column #1). Therefore, functionality of part BBa_K2959010 as a generator of AtPFN1 under induction by IPTG was confirmed.
 
As shown in figure 3, bands are presented at the approximate weight of 14.3 kDa which corresponds to AtPFN1 as corroborated by UniProt. Visible bands of AtPFN1 correspond to protein extracts of transformed <i>E. coli</i> BL21 (DE3) induced with different concentrations of IPTG. No band can be appreciated in the negative control column, following the protein ladder, which belongs to a control of untransformed cells, afirming that the bands are indeed AtPFN1. No basal level expression was observed on the uninduced control (transformed cells with no IPTG, column #1). Therefore, functionality of part BBa_K2959010 as a generator of AtPFN1 under induction by IPTG was confirmed.
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===Antifungal Assay===
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In order to test the antifungal activity of our peptides as well as prove their viability as a mechanism to inhibit <i>Verticillium dahliae</i>, an antifungal susceptibility test on a 96 well plate was carried out by measuring absorbance at 405 nm, wavelength used in standardized protocols to measure growth of filamentous fungi<sup>3</sup>. Due to a lack of time, we couldn’t reach the experimental stage of the project of peptide purification, so experiments were made using soluble protein extracts from our transformed cells’ lysates. Different dilutions of the extracts were prepared which were applied to a spore suspension of <i>V. dahliae</i>. Dilutions of the extracts of untransformed cells were used as controls to prove that inhibition was the result of the peptides and not any other protein contained within the extract. Soluble proteins from <i>E. coli</i> BL21 (DE3) were used as control for the AtPFN1 extract. Concentrations of extracts with peptides were equalized to their controls. Table 1 details the concentrations of each dilution.
 
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Revision as of 23:01, 21 October 2019

Expressible Arabidopsis thaliana Profilin 1

This composite part consists of a T7 promoter, ribosome binding site, a coding sequence for AtPFN1 as a fusion protein with a 6x His-Tag, and a double terminator. This construct allows the expression of AtPFN, an antifungal peptide from Arabidopsis thaliana in E. coli BL21 (DE3). Expression can be positively regulates by the addition of IPTG thanks to its promoter. The part is designed to code for a fusion protein of AtPFN1 with a polyhistidine tag (6x His-Tag) at its C-terminus for purification by immobilized metal affinity chromatography.


Usage and Biology

AtPFN1 is a protein extracted from the plant Arabidopsis thaliana, it is a profillin which means that is an actin binding protein and weights 14 kDa1. It inhibits fungal cells growth by penetrating the cell wall and membrane, generating reactive oxygen species and mitochondrial superoxide triggering cell apoptosis, resulting in morphological changes in the cells2.

The binding affinity of antifungal proteins to fungal cells is the most important attribute for their fungal action, even if the mechanism is membranolytic or cell damaging. It has also been demonstrated that these proteins can be transferred across the cell membrane into the cytosolic space and accumulate in the cytosol of the cell by altering the membrane integrity. For cytosolic translocation the mechanisms used by the proteins are direct penetration, vacuolar localization and expansion, partial plasma membrane disruption, transition pore formation and endocytosis. AtPFN1 has exhibited a potent antifungal activity against fungal strains of C. gloesporioides, F. osysporum, C. albicans, and C. glabrata2.


Characterization of Expressible Arabidopsis thaliana Profilin 1

Our DNA sequence AtPFN1, was synthesized by IDT®️ with the Biobrick prefix and suffix flanking the composite part. This made possible the correct digestion with restriction enzymes EcoRI-HF and PstI. After the digestion, ligation was performed with T7 ligase in order to place our construct into the pSB1C3 linearized backbone with chloramphenicol resistance, which was previously digested with the same restriction enzymes. Using the SnapGene®️ software, we could model our ligated expression plasmid, and the final parts resulted in a sequence of 2,651bp. Thereupon, Escherichia coli BL21(DE3) was transformed by heat shock for following antibiotic selection of clones.

T--Tec-Chihuahua--AtPFN1ff.png
Figure 1. SnapGene®️ map of BioBrick BBa_K2959010.


The next step was to amplify our BioBrick sequence through colony PCR performed upon our transformed cells to confirm the presence of our expression plasmid inside of our chassis. With the help of the specific forward Biobrick prefix [BBa_G1004] and the specific reverse Biobrick suffix [BBa_G1005], we were able to amplify our sequence exclusively. Through an agarose gel we confirmed the correct transformation. The PCR action from SnapGene®️ was used to predict the size of the amplified sequence which resulted in a size of 640 bp.

T--Tec-Chihuahua--atpfn.pcr.png T--Tec-Chihuahua--ELECTROFORESISAtPFN.jpeg
Figure 2. (On the left) SnapGene®️ amplification through PCR of BBa_K2959010. (On the right) Agarose gel electrophoresis of BBa_K2959010 compared with NEB Quick-Load Purple 1Kb Plus DNA Ladder, where the highlighted band corresponds to approximately 640 bp.


Protein production

IPTG Induction and Extraction

Following the construction of the BioBrick, it was necessary to induce protein production. Since T7 promoter for AtPFN1 is used due its high levels of transcription in E.coli BL21 (DE3), isopropyl β-D-1 thiogalactopyranoside (IPTG) is employed as an inducer for T7 RNA polymerase production. The concentration of IPTG utilized was 0.2 mM, 0.4mM, 1mM and 3mM, followed by incubating the cultures at 37°C and 225 rpm for 5 hours. This was followed by protein extraction by lysis solution to which lysozyme was added, in order to obtain our soluble peptides.


SDS-PAGE

Electrophoresis in a 12% polyacrylamide gel was performed in order to corroborate that the protein of interest was indeed expressed. Each well was loaded with 50 μg of total protein from the soluble extracts of the cell lysates.

T--Tec-Chihuahua--Gel.jpeg
Figure 3. SDS-PAGE (12%) of E. coli BL21 (DE3) transformed with BBa_ K2959010. (Negative control: E.coli SHuffle untransformed cells, 1. ATPFN1 transformed cells without induction, 2. ATFPN1 transformed cells with 0.2mM of IPTG, 3. ATFPN1 transformed cells with 0.4mM of IPTG, 4. ATFPN1 transformed cells with 1mM of IPTG, 5. ATFPN1 transformed cells with 3mM of IPTG, 6. ATFPN1 transformed cells with 3mM of IPTG)

As shown in figure 3, bands are presented at the approximate weight of 14.3 kDa which corresponds to AtPFN1 as corroborated by UniProt. Visible bands of AtPFN1 correspond to protein extracts of transformed E. coli BL21 (DE3) induced with different concentrations of IPTG. No band can be appreciated in the negative control column, following the protein ladder, which belongs to a control of untransformed cells, afirming that the bands are indeed AtPFN1. No basal level expression was observed on the uninduced control (transformed cells with no IPTG, column #1). Therefore, functionality of part BBa_K2959010 as a generator of AtPFN1 under induction by IPTG was confirmed.

Antifungal Assay


In order to test the antifungal activity of our peptides as well as prove their viability as a mechanism to inhibit Verticillium dahliae, an antifungal susceptibility test on a 96 well plate was carried out by measuring absorbance at 405 nm, wavelength used in standardized protocols to measure growth of filamentous fungi3. Due to a lack of time, we couldn’t reach the experimental stage of the project of peptide purification, so experiments were made using soluble protein extracts from our transformed cells’ lysates. Different dilutions of the extracts were prepared which were applied to a spore suspension of V. dahliae. Dilutions of the extracts of untransformed cells were used as controls to prove that inhibition was the result of the peptides and not any other protein contained within the extract. Soluble proteins from E. coli BL21 (DE3) were used as control for the AtPFN1 extract. Concentrations of extracts with peptides were equalized to their controls. Table 1 details the concentrations of each dilution.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


References

1. Christensen, H. E. M., Ramachandran, S., Tan, C.-T., Surana, U., Dong, C.-H., & Chua, N.-H. (1996). Arabidopsis profilins are functionally similar to yeast profilins: identification of a vascular bundle-specific profilin and a pollen-specific profilin. The Plant Journal, 10(2), 269–279. doi:10.1046/j.1365-313x.1996.10020269.x
2. Park, S. C., Kim, I. R., Kim, J. Y., Lee, Y., Kim, E. J., Jung, J. H., ... & Lee, J. R. (2018). Molecular mechanism of Arabidopsis thaliana profilins as antifungal proteins. Biochimica et Biophysica Acta (BBA)-General Subjects, 1862(12), 2545-2554. doi:10.1016/j.bbagen.2018.07.028