Team:ACIBADEM ISTANBUL/Measurement

Our parts

We designed 3 different parts, and inserted into pPIC9K vector to express in Pichia pastoris. Our parts are;

http://parts.igem.org/Part:BBa_K3272003

http://parts.igem.org/Part:BBa_K3272000

Why we used Pichia pastoris?

Efficient transormation to Pichia postoris is a challenging process and need a long-term optimization.
As high amounts of DNA is required for efficient transformation, we first amplified our parts using E.coli.
To obtain the best clones we tried each of the part we desinged and repeated inoculation till we get single clones on the plates.

Expression vector pPIC9K

Our system improved our team's previous iGEM project (Team ACIBADEM_ISTANBUL, 2018):
Last year, the main issue we faced was the transormation and exression efficiency and the production cost of out protein.
This year, using pPIC9K instead of pPICZAlpha A vector was much more cost effective because we don't have to use expensive zeocin antibiotic.
Instead, we used Kanamycin and Ampicillin antibiotics to select the clones.

How we purify our protein?

Debugging

We encountered one slight problem with the proteins that were delivered by Peptide 2.0, that being a large amount of electrostatic activity causing the lyopholized peptides to stick to our weighing spatula. This resulted in us having to wash off the particles stuck to the weighing spatula back into their containers, lyophilzing it (a process that takes 24 hours) just to be able to weigh them for our experiments. Sean Fisher from Peptide 2.0’s Customer Service stated that the electrostatic activity is most likely due to the peptides interaction with the plastic containers they were sent with. As a solution, they have agreed to aliquot our future peptide orders into several vials in order to avoid this problem.

Wet Lab

Stability test by HPLC

According to our results, C_LTNF_C and C_LTNF_C without stop codon are more stable in blood stream compared to the native LTNF 10:

Hemolytic Activity Assay

According to our results, C_LTNF_C and C_LTNF_C without stop codon does not have significant haemorrhaegic effect, even at higher doses:

Dry Lab

Homology Modelling

For the in silico homology modelling I-Tasser program was used. Since, there wasn’t any reference template for our protein, homology modelling was done by similarity. However, confidince scores’ of the protein were very low. LTNF10 native and C_LTNF_C were modelled.


Fig.1. LT10 homology model (c –score : -0.55)	Fig.2. C_LTNF_C homology model (c-score : -2.44)

Since the modelling scores were very low, repair process was applied to the proteins via FOLDX program. A function, RepairPDB, repaired the models and tried to lower their energy.

Modification PDB

The way it operates is the following: First it looks for all Asn, Gln and His residues and flips them by 180 degrees. This is done to prevent incorrect rotamer assignment in the structure due to the fact that the electron density of Asn and Gln carboxamide groups is almost symmetrical and the correct placement can only be discerned by calculating the interactions with the surrounding atoms. The same applies to His. We do a small optimization of the side chains to eliminate small VanderWaals’ clashes. This way we will prevent moving side chains in the final step. We identify the residues that have very bad energies and we mutate them and their neighbours to themselves exploring different rotamer combinations to find new energy minima. FoldX uses output-file as a tag to label different outputs from different commands in batch runs.