Team:Lund/Results

iGEM Lund 2019

Results and Discussion

This is the page were we present our results.

Overview

The laboratory experiments were started with the restriction cloning of the parts with the pET11a vector and followed with heat shock transformation in TG1 cells. The successful cloning was confirmed primarily using colony PCR and later by Sanger sequencing. The plasmids were transformed to BL21 DE3 and were used in protein expression. The expressed proteins were identified using SDS-PAGE. The protocols followed in each experiment are detailed in the Protocol and Experiments

The following eight parts were successfully transformed into E. coli BL21 (DE3):

Protein expression was successfully performed for all these parts except Trx-Ec-AMP-D1.

Parts

Several basic parts were added to the registry and 17 composite parts were designed to express AMPs and the protease enterokinase as fusion proteins. For more information about the design of the parts see Project Design, and for a list of the basic and composite parts used see the Parts page.

Transformation and Isolation of BL21 (DE3):

The cloned pET11a plasmids containing the antimicrobial peptide sequences were transformed into TG1 competent cells by heat shock transformation method. Five single colonies were picked up from each of the agar plates having the transformants. These colonies were used for preparing colony PCR as well as master plate. The gel image of the sample obtained from the PCR colony is given in Figure 1.

Figure 1: Gel showing the results of colony PCR with the 100 bp DNA ladder and Thioredoxin (Trx). The band marked with red arrow are the samples which showed 100% sequence identity in sanger sequencing and were further transformed into BL21 (DE3).

Sequencing of Composite parts:

Plasmid purification was made for the transformants and three samples of each of the eight inserts were sent to Eurofins Genomics for sanger sequencing. Four samples of each insert for the first batch of composite parts were sequenced and all had at least one sample of 100% sequence identity with the constructed parts, see analysed data here and raw data here. This proves that the correct sequences have been transformed for composite parts presented in table 1.

Insert Parts Number of transformants with 100% identity
Trx-EAFP1 BBa_K3619014 1
Trx-Ec-AMP-D1 BBa_K3619015 2
Trx-MsrA2 BBa_K3619016 3
Trx-nmDEF02 BBa_K3619018 3
Trx-Rs-AFP1 BBa_K3619017 2
Trx-Temporin A BBa_K3619020 3
GST-Scarabaecin BBa_K3619028 3
Thioredoxin BBa_K3619031 3

Cultivation and Protein expression:

The 8 constructs which were successfully transformed into the BL21 (DE3) cells were cultivated in TB media and the proteins were expressed with the induction of 1mM IPTG.

The growth curve (figure 2) depicts that all the transformed cells except for Trx- EAFP 1, are in their log phase until roughly 6 hours after induction and then enter the stationary phase. Whereas Trx- EAFP 1 continues to follow the log phase. So the E. coli cells accepted our AMP gene and were able to reproduce successfully without our AMPs inhibiting its metabolic processes.

Figure 2: the growth curve of the BL21 (DE3) cells after the induction of IPTG.

SDS-page samples of the cultivation were taken every 2 hours after IPTG induction and protein expressions were seen in all hour samples but the 8 hours samples (late log phase) showed a higher amount of protein comparatively when visualized through SDS-page. From the total of 8 constructs we successfully transformed, one was Thioredoxin and the other seven were our AMPs with Thioredoxin as fusion partner. Six of our seven AMPs showed very good desired protein expression, see figure 3. This means that E. coli was a very good expression system and also our gene design for the different AMPs worked well and can be repeated for future works.

Insert Trx-Temporin A Trx-Msr A2 Trx-NmDEF02 Gst-Scarabaecin Trx-Ec-AMP-01 Trx-EAFP1 Trx-Rs-AFP1 Thioredoxin
Size (kDa) 14.73 16.63 18.47 28.47 18.21 17.56 19.03 13.35

Figure 3. SDS PAGE gel showing the proteins obtained from the 8 constructs after 8 hours after IPTG induction and the control sample with its respective ladder size.

Conclusion

We successfully designed and expressed almost all of the proteins with Thioredoxin as a fusion partner as well as one construct with GST as a fusion partner in E. coli BL21 and the results are promising. We also optimised the expression time and determined it to be 8 hours after induction of IPTG.

Future plans

In the future, more testing needs to be conducted in order to show the effectiveness of the different peptides, and to be able to decide on the most potent combination of peptides towards treating late blight. We plan on first testing the cell lysate on isolated P. infestans, and after evaluating the effectiveness of the peptides, we would continue with the most promising ones and test them on potato leaves, to better simulate reality. We would also test the peptides on whole potato plants to simulate as natural an environment as possible. This would enable us to conduct tests during a longer period of time and see if there is any harm to the plant from the lysate. We would also be able to test how often the cell lysate has to be reapplied and how environmental conditions such as rain affects the application frequency.

As of now, we transformed but neither sequenced nor expressed the constructs with GST as a fusion partner, but this is something we would like to do. Scarabaecin fused to GST showed very good expression, so more experiments with GST should be done before finally deciding on which fusion partner is to be used.

When having decided on the most potent mixture of peptides and the most suitable fusion partner, we plan on trying the whole two plasmid-system with Enterokinase in one plasmid and the peptides in another plasmid. Making this work, with Enterokinase cleaving away the fusion partner and separating the different peptides, would be critical to obtain an economically viable product. The kill switch would also have to be tested in our system, to ensure that the product is completely safe to the environment and that no living GMMs are released.

We did not have time to try the peptides WHIPR and MAFP-1 that the model created, but gaining some experimental data could greatly improve and validate the model. By having a model that can continuously come up with effective antifungal peptides, the problem of P. infestans becoming resistant to the peptide mixture would be avoided since one or more of the peptides could be switched to another one suggested by the model.

In conclusion, we have gained positive results so far and would be able to continue developing the project.