Engineering Success
Engineering Design Cycle #1: Destructin-1 Full Sequence
In order to test our final intended product for our project, an inhibitor to the mature destructin-1 protein that has enzymatic function, we need to have the inactive form of the protein. The destructin-1 full sequence contains both the mature protein sequence as well as the leader peptide sequence, rendering the protein inactive.
To make the comparison to see if we have inactivated the mature protein, we will need data to compare it to. Thus, we are attempting to transform the full destructin-1 sequence into E.Coli for amplification and analysis. These attempts have currently proven to be unsuccessful.
Design
The intended function of the Destructin-1 full protein is to be used for comparison. The sequence for Destructin-1 was ordered with its intended use being to be inserted into E.coli for amplification and comparison. Engineering principles were used to specify this biological system with the intended function of providing a control for our inhibitor testing.
Build
The sequence was transformed into E.coli for amplification and analysis, then grown in LB media and LB agar plates. The desired DNA sequence encoding the biological system was constructed and introduced to our chassis organism.
Test
After the transformation was grown up in E.coli, a mini-prep was run to isolate the desired sequence. Once the mini-prep was finished, the resulting purification was analyzed via DNA Gel Electrophoresis alongside the original sequence used for the transformation. The function of the engineered biological system was assayed through molecular analysis.
Learn
The transformed sequence purification did not match the original sequence once analyzed on a gel. There were additional, unintended sequences present in the gel. The discrepancies between the desired and observed function were analyzed with quantitative data. Further isolations may have to be made to ensure that only the intended sequence is achieved through transformation. Attempts for this could be adding an antibacterial resistance marker for Kanamycin to ensure that only cells with the intended sequence can proliferate.
Figure 1: Dst1 full sequence transformation growth on an LB Agar plate
Figure 2: Dst1 full sequence transformation gel
Lane 1: DNA Ladder
Lane 2: Transformed Dst1 full sequence
Lane 3: Intended sequence
Engineering Design Cycle #2
Since the previous engineering cycle was unsuccessful in expressing the full Destructin-1 protein in E. coli, a second cycle was initiated. This cycle builds on insights gained from earlier attempts, with newly designed constructs and a revised workflow outlined below:
Design
New constructs were made containing the mature destructin-1 protein or the destructin-1 leader peptide alone, each with a c-terminal hexa-histidine tag attached to a short GSG linker.
Build
The constructs were assembled with the MalE E. coli maltose-binding protein periplasmic secretion signal and the Arabad arabinose-inducible promoter and cloned into an in-house vector containing a terminator.
Test
E. coli cells were transformed with destructin-1 protease or leader peptide constructs and confirmed by PCR. Test expressions were performed by growing up colonies overnight in 1mL cultures with Arabinose for induction of protein expression. An SDS-PAGE was run to assess protein expression levels.
Learn
A faint band at the expected molecular weight for the leader peptide was observed, suggesting possible expression, but expression levels do not appear to be very high. Affinity purification will be necessary to confirm the authenticity of the band.
The mature destructin pellet formed an insoluble pellet that could not be loaded onto the gel – this may be due to the action of the protease. A very small amount of protein is visible in the appropriate lane, and there may be a band corresponding to the destructin protein. This may or may not be a problem when we attempt to purify the protein, or we may need to express the pre-protein and attempt to activate it in-vitro for further work.
Figure 3: Gel of the empty vector, control protein, destructin leader peptide, and mature destructin highlighting possible correct bands.
Conclusion for Both Cycles
These results show that our project is progressing in the right direction. While expression of the Destructin-1 constructs in E. coli remains challenging, these issues could be addressed through further engineering cycles. Future efforts will focus on improved isolation, additional purification strategies, and potentially expressing the pre-protein followed by in vitro activation. These steps will allow us to achieve reliable expression of Destructin-1 in E. coli, which would enable us to establish a baseline for evaluating the effectiveness of our inhibitor.
Documenting a Design Cycle
Design: Engineering principles are used to specify a biological system with an intended function, and models are used to help improve the initial design according to these principles.
Build: The desired DNA sequence encoding the biological system or a part of the biological system is constructed and introduced to a chassis, frequently the target organism.
Test: The function of the engineered biological system is assayed.
Learn: The discrepancies between the desired and observed function are analyzed, frequently with quantitative data, to develop improved models and design parameters.
References
- A.J. et al. (2015) 'Destructin-1 is a collagen-degrading endopeptidase secreted by Pseudogymnoascus Destructans, the causative agent of white-nose syndrome', Proceedings of the National Academy of Sciences, 112(24), pp. 7478–7483. doi:10.1073/pnas.1507082112.
- S., Holland, I.B. and Schmitt, L. (2014) 'The type 1 secretion pathway — the hemolysin system and beyond', Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1843(8), pp. 1629–1641. doi:10.1016/j.bbamcr.2013.09.017.
- T., Chaturvedi, V. and Chaturvedi, S. (2015) 'Novel trichoderma polysporum strain for the biocontrol of Pseudogymnoascus destructans, the fungal etiologic agent of Bat White-nose syndrome', PLOS ONE, 10(10). doi:10.1371/journal.pone.0141316.