Results

Achievements

The construction of five new BioBricks™ with sequences that are reported to code for our proteins of interest:

The correct transformation of E. coli expression strain SHuffle T7 Express®️ for parts BBa_K2959005 (PsDef1- Erv1p) and BBa_K2959007 (WAMP1b); and the correct transformation of BL21 (DE3) Competent E. coli for part BBa_K2959010 (AtPFN1).
Protein production characterized in part BBa_R0010, a lacI regulated promoter, in E. coli expression strain SHuffle T7 Express®️, confirming IPTG induction functionality in our parts.
The amplification of our BioBrick™ sequences through colony PCR performed upon our transformed cells to confirm the presence of our expression plasmids.
Corroboration of protein expression in E. coli BL21 (DE3) transformed with BBa_ K2959010 through an SDS- PAGE at 12%.
The antifungal activity was proved with the three peptides: AtPFN1 [BBa_K2959010],PsDef1 [BBa_K2959005],WAMP1b [BBa_K2959007] against V.dahliae.

BioBrick™ Assembly

Three of our DNA sequences (AtPFN1, PsDef1-Erv1p, and WAMP1b) were 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 constructs 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 plasmids, and the final parts resulted in a sequence of 2,590 bp for WAMP1b, 2,651bp for AtPFN1 and 3,106 bp for PsDef1-Erv1p. Thereupon, Escherichia coli [BL21(DE3) for AtPFN1 and SHuffle for PsDef1+Erv1p and WAMP1b] cultures were transformed by heat shock for following antibiotic selection of clones.

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Figure 1. SnapGene®️ maps of a) BioBrick™ BBa_K2959007, b) BioBrick™ BBa_K2959010, and c) BioBrick™ BBa_K2959005 ligated into backbone pSB1C3.

The next step was to amplify our BioBrick™ sequences through colony PCR performed upon our transformed cells to confirm the presence of our expression plasmids 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 sequences exclusively. Through agarose gels we confirmed the correct transformation. The PCR action from SnapGene®️ was used to predict the size of the amplified sequences which resulted in a size of 579 bp for WAMP1b, 640 bp for AtPFN1, and 1,095 bp for PsDef1+Erv1p.

Figure 2. (On top) SnapGene®️ amplification through PCR of BBa_K2959007. (Left) Agarose gel electrophoresis of BBa_K2959007 compared with NEB Quick-Load Purple 1Kb Plus DNA Ladder, where the highlighted band corresponds to approximately 579 bp.

Figure 3. (On top) SnapGene®️ amplification through PCR of BBa_K2959010. (Left) 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.

Figure 4. (On top) SnapGene®️ amplification through PCR of BBa_K2959005. (Left) Agarose gel electrophoresis of BBa_K2959005 compared with NEB Quick-Load Purple 1Kb Plus DNA Ladder, where the highlighted band corresponds to approximately 1,095 bp.

Protein Production

IPTG Induction and Extraction

Following the construction of each 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, followed by incubating the cultures at 37°C and 225 rpm for 5 hours. Production of PsDef1 and WAMP1b was induced under the lacI regulated promoter in E. coli SHuffle cells with IPTG 0.2 mM and incubated at 30°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 (Figure 5) 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.

Figure 5. Expression of AtPFN1 visualized in a SDS-PAGE (12%) of E. coli BL21 (DE3) transformed with BBa_K2959010 (Negative control: E.coli BL21 (DE3) 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, bands are present 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 first column following the protein ladder Precision Plus Protein^TM Dual Xtra Prestrained Protein Standars, which belongs to a control of untransformed BL21 (DE3) cells, confirming that the bands are expressed exclusively in transformed cells and are indeed AtPFN1. No basal level expression was observed on the uninduced control (transformed cells with no IPTG) as seen in column 1. Therefore, functionality of part BBa_K2959010 as a generator of AtPFN1 under induction by IPTG was confirmed.

For parts BBa_K2959005 and BBa_K2959007, visualization of PsDef1 and WAMP1b in a polyacrylamide gel could not be carried out given the small size of the peptides, 9 and 4.5 kDa respectively. Due to a lack of reagents and equipment, we were unable to adapt our SDS-PAGE protocol and optimize the visualization of small proteins. However, to confirm that our peptides were being expressed, we characterized the lacI regulated promoter (BBa_R0010), which controls expression of these two parts, in E. coli SHuffle. The functionality of this promoter as well as ideal IPTG concentrations and incubation temperatures were confirmed. Thus, BBa_K2959005 and BBa_K2959007 work as generators of PsDef1 and WAMP1b when induced with 0.2 or 0.4 mM IPTG at 30°C. Read more about it in our Characterizationpage.

Demonstrate

In addition to the molecular characterization, we decided to characterize our parts in a more realistic environment by confronting them against the fungus. For this reason, we decided to set up a test on a microplate where the confrontation of our peptides adjacent to V.dahliae took place. Here, peptides’ inhibition capability was detected through a growth curve of the fungus. Derived from this, we could achieve positive results where two or more concentrations of our peptides (raw extraction) showed some kind of inhibition: for more info click the button below.

Successes & Failures

During our iGEM experience, we learned a lot of unique things that led to significant achievements after facing unexpected obstacles. After months of hard work, we believe the effort was worthwhile.

Successes

The antifungal activity was proved with the three peptides: AtPFN1 [BBa_K2959010], PsDef1 [BBa_K2959005], WAMP1b [BBa_K2959007] against V.dahliae.
Characterization of LacI regulated promoter (BBa_R0010) and RFP Coding Device (BBa_J04450).
Protein production characterized with a LacI promoter (BBa_R0010) in E. coli SHuffle T7 Express®️, confirming IPTG induction functionality in our parts.
We achieved the assembly of three new BioBricks. BBa_K2959005 that codes for PsDef1-Erv1p, BBa_K2959007 that codes for WAMP1b. BBa_K2959010 that codes for AtPFN1. The three of them are expression systems for antimicrobial peptides.

Unsuccessful

We first started to work with E. coli DH5α but later had to change to Mach1 due to the small colonies we achieved during transformation.
We also discovered that one of the strains we thought was PsDef1 didn’t actually have the protein. After transformation, we saw colonies and assumed that it had been correctly transformed. Electrophoresis was performed and it showed the band of the whole construct. After working with that construct for a long time, we performed electrophoresis again only to see that the band of the size of pSB1C3 was observed but not the rest, it just vanished, and we are not sure where it vanished while we were working. Up to this day, we are not sure how it disappeared.
SHuffle T7 Express®️ was the most important chassis of two of our parts and they were correctly transformed for the first half of summer. The transformation and competent cells protocol were placed aside, and we didn’t use it for a while since we were working on other parts of the project. When we used SHuffle in order to transform other parts, the strain didn’t work properly. The LB - CAM plates disseminated with control competent cells, showed colony growth and this was odd for us. Our LB + CAM plates didn’t show any growth, which meant we didn’t have any contamination and our RFP had grown but the colonies weren’t red. We got to the conclusion that our competent cells weren’t completely competent anymore, that they had lost this characteristic due to the amount of time it was stored at -70ºC.

Future Plans

For future experimentation we have the following plans to impulse our project:

Team:Tec-Chihuahua/Results