Laboratory Safety
Safety is an important aspect of researching. Before designing our experiment, we have to prepare
and take countermeasures by making biosafety analysis of our project. Microorganism we used in
our project is E. coli K-12 strain which is categorized as WHO Risk Group 1. We are
working in Biosafety Level 1 laboratory, Virology and Cancer Pathobiology Research Center (VCPRC),
Faculty of Medicine, University of Indonesia which has been certified internationally by the
World Health Organization, European Committee of Standardization, and World BioHazTec
Before working in VCPRC laboratory we have to meet safety criteria such as risk assessment of
each project, biosafety training, laboratory orientation, and understanding all the standard
operational procedure.(Figure 2) There are also protective gear to ensure the safety of
the lab member.(Figure 1) The temperature and pressure of the lab is always maintain to
make sure there are no airborne or gas leak out of the lab. VCPRC safety quality control is Mrs.
Aroem Naroeni as the biosafety officer certified by International Federation of Biosafety
Associations.
Our project used diphtheria toxin; hence, to make sure the safety of the lab member, we created a
modified diphtheria toxin, DiphTox. We modified the toxin by just using the toxic region and
maintaining the ability to bind. Our modified diphtheria toxin has been checked by: (1)
Dr.dr.Budiman Bela Sp.MK(K), our principal investigator; (2) Mrs. Aroem Naroeni, biosafety
officer in Virology and Cancer Pathobiology Research Center (VCPRC); (3) Biosafety and
Biosecurity Committee of IGEM 2019; and (4) Michelle E. Rozo, PhD from Biosecurity Engagement
Programme, U.S. Department of State - which meeting was conducted in the Embassy of the United
States of America, Jakarta-Indonesia. (Figure 3)
Diphtheria Toxin Safety Analysis
Most of this safety analysis was performed by iGEM UI_Indonesia 2018. We added a
few analysis to ensure further safety of the toxin. To ensure the affitoxin has no toxicity we
perform literature search. First we need to know every domain that construct whole Diphtheria
Toxin (DT). We are using online databases such as UNIPROTand INTERPRO.1,2
We found that DT consists of 3 domains (Figure 4), which every each of them has specific
function.
DT is classified to AB toxin which A fragment consists of C (catalytic) domain and b fragment
consists of T (translocation) and R (recognition) domain (Figure 5).3
During the intoxication DT binds to proHBEGF on surface membrane. EGF like domain on proHBEGF
receptor bind the R domain of DT. Receptor bound toxin is concentrated in clathrin coated pits
and internalized to clathrin coated vesicle. With the increase of vacuolar ATPase activity, the
pH inside vesicle is decreased. The decrease of pH will change the conformational structure of
DT, especially T domain. T domain function is to translocate C domain to cytosol. There are many
hypotheses about the mechanism of translocation C domain from lumen to cytosolic space. After C
domain delivered to cytosol, it changes in structure into enzymatically active conformation and
catalyzes the NAD-dependent ADP ribosylation of EF-2 which leads to cellular death (apoptosis)
via protein synthesis inhibition. One publication said that only A substance can lead to
cellular death. The mechanism of intoxication is summarized in Figure 3.4,5
From this theory we conclude that A fragment or C domain is the main cause of cytotoxic activity.
T and R domain function is only for delivery of C domain into cytosol. In order to maximize the
safety we want to take as small as we can but still retain the binding activity of DT.
Publication by John M. Rolf and Leon Eidels said that last 54 amino acids (482-535) are
sufficient to make HT and HBEGF bind. These 54 amino acids is part of R domain which has no
cytotoxicity effect.6
