Overview #

For our so­lu­tion to be truly ben­e­fi­cial to so­ci­ety, it is crit­i­cal for it to be safe. Here at IISER Bhopal, we have put im­mense im­por­tance on our pro­jec­t’s biosafety and ethics, in­spected our work at var­i­ous stages and at­tempted to cover every as­pect. This sec­tion di­vides the safety con­sid­er­a­tions into Design, Laboratory Conduct, Experimental Safety, and Human Subject Ethics.

Design #

Kill Switch #

Since we plan to build an on-field mi­cro­bial spray from our en­gi­neered bac­te­ria (see fu­ture im­ple­men­ta­tion) sim­i­lar to the ex­ist­ing de­com­posers con­sist­ing of lyophilized mi­crobes, it is es­sen­tial to con­sider the ef­fects it can have on the nat­ural soil mi­cro­biota pre­sent in the agri­cul­tural fields.

The na­tive mi­cro­bial pop­u­la­tions pre­sent in the soil con­sist of es­sen­tial fun­gal and bac­te­r­ial species that have a di­verse set of func­tions, from re­cy­cling min­er­als, form­ing soil crys­tals through bio­min­er­al­iza­tion, act­ing as an un­tapped re­source for an­tibi­otic dis­cov­ery, and even fa­cil­i­tat­ing plant-plant com­mu­ni­ca­tion for re­silience against dis­eases and en­vi­ron­men­tal stres­sors [^1]. Hence, it is es­sen­tial for our so­lu­tion to cre­ate min­i­mal dis­tur­bance in this sys­tem.

To en­sure that our bac­te­ria get cleared off the field once the de­com­po­si­tion is achieved, we have the­o­ret­i­cally de­signed a kill switch in­volv­ing an AND gate be­tween the L-arabinose in­ducible pro­moter pBAD and D-xylose in­ducible pro­moter pXyl (BBa_K851002, sub­mit­ted by (GEM12_UNAM_Genomics_Mexico). L-arabinose and D-xylose are re­leased from the degra­da­tion of hemi­cel­lu­lose (arabinoxylan), which forms an av­er­age of 25–35% of crop stub­ble [^2]. From the lit­er­a­ture, we found out that the per­cent­age di­gestibil­ity of hemi­cel­lu­loses from the Xylan fam­ily is lower than that of Celluloses. This led us to choose the above given reg­u­la­tory sys­tem, which will be able to sense the break­down prod­ucts of the last poly­mers un­der­go­ing di­ges­tion.

Downstream to this reg­u­la­tory sys­tem, we have added yqcG (BBa_K3507002, sub­mit­ted by iGEM20_­Gronin­gen), which codes for a po­tent DNAse pro­tein [^3]. YqcG is a part of the Type II (protein-protein) toxin-an­ti­toxin sys­tem YqcG/F found in Bacillus sub­tilis. It is in­volved in caus­ing pro­grammed cell death in biofilms by frag­ment­ing the chro­mo­so­mal DNA of the bac­te­ria. eGFP (enhanced GFP) is the re­porter added to the cir­cuit which ends with a ter­mi­na­tor (Ter) cod­ing for two stop codons (taataa).

Kill Switch Circuit #

pBAD-pXyl AND Gate #

The first two sites rep­re­sent the pBAD-AraC reg­u­la­tory re­gions, where the AraC pro­tein in­her­ently pre­sent in B. sub­tilis binds to neg­a­tively reg­u­late down­stream gene ex­pres­sion.

The SigmaA -35, Sigma A-10, and the XylR op­er­a­tor re­gions rep­re­sent the bind­ing and in­ter­ac­tion sites for tran­scrip­tion reg­u­la­tor B.subtilis XylR, which de­tects the pres­ence of D-Xylose.

Specifications of each part #

pBAD:

• Upstream reg­u­la­tory sys­tem con­sists of two ara bind­ing sites that in­ter­act with a pro­tein dimer AraC. AraC causes the DNA to loop in the ab­sence of L-arabinose
• When suf­fi­cient ara­bi­nose con­cen­tra­tions are reached, AraC changes con­for­ma­tion abruptly, straight­en­ing the DNA and mak­ing the down­stream cod­ing re­gion ac­ces­si­ble.
• It is very tightly reg­u­lated and non-leaky, hence ideal for use with down­stream tox­ins.

More de­tails: http://​parts.igem.org/​Part:BBa_K851002

Reporter #

eGFP (enhanced GFP, BBa_K1915001 by iGEM16_Lam­bertGA) is the re­porter added to the cir­cuit which ends with a dou­ble-stop codon (taataa) se­quence as the ter­mi­na­tor (Ter).

Future Implications #

Throughout the course of our pro­ject, we got nu­mer­ous op­por­tu­ni­ties to dis­cuss the biosafety is­sues per­tain­ing to our goal with ex­perts in the field through meet­ings and col­lab­o­ra­tions. We have given a gist of the con­clu­sions we could de­rive.

Laboratory Conduct #

Safety Cell at IISER-Bhopal The Safety Office at Indian Institute of Science Education and Research, Bhopal is com­mit­ted to pro­vide a safe and healthy en­vi­ron­ment at its cam­pus. Our team fol­lowed the rules and guide­lines es­tab­lished by our in­sti­tu­tion for the use of equip­ment and reagents, waste dis­posal, and san­i­ti­za­tion. We had re­ceived train­ing on safe lab con­duct as well as in­struc­tions on how to use equip­ment and ma­te­ri­als for ba­sic mi­cro­bi­ol­ogy and syn­thetic bi­ol­ogy ex­per­i­ments as part of our uni­ver­sity course­work. Our ex­per­i­ments in the lab­o­ra­tory were fre­quently per­formed un­der su­per­vi­sion of lab­o­ra­tory tech­ni­cians, and we were trained rig­or­ously on the proper ex­e­cu­tion of the pro­to­cols used through­out the pe­riod of the pro­ject.

Institute level check­list to en­sure ba­sic lab safety: https://​www.iis­erb.ac.in/​as­sets/​al­l_u­p­load/​safety/​Std_Lab_Safe­ty_Check­_List.pdf

National level safety guide­lines: https://​dbtin­dia.gov.in/​sites/​de­fault/​files/​up­load­files/​Reg­u­la­tions_%26_Guide­lines_­for_Re­ocmi­nan­t_D­NA_Re­search_and_Bio­con­tain­ment%2C2017.pdf

Experimentation #

The ex­per­i­ments were car­ried out at the BSL-1 Undergraduate lab­o­ra­tory at our home in­sti­tute. We pri­mar­ily used E.coli DH5⍺ and Bacillus sub­tilis 168 as our chas­sis or­gan­isms, which are char­ac­ter­ized un­der Risk Group 1 with neg­li­gi­ble haz­ardous ef­fects. These or­gan­isms are known to be non-path­o­genic in hu­mans and plants, mak­ing them safe to work with in the BSL-1 lab­o­ra­tory while fol­low­ing ba­sic lab con­duct.

Moreover, proper lab­o­ra­tory tech­niques were used when work­ing with these strains and our team en­sured to use proper PPE such as gloves, lab coats, and closed-toe shoes while work­ing in the lab­o­ra­tory. The mi­croor­gan­isms were con­sis­tently han­dled un­der Laminar air flow cab­i­nets, and the work­spaces were san­i­tized reg­u­larly with Ethanol and UV. Our lab­o­ra­tory ex­per­i­ments were fre­quently per­formed un­der the su­per­vi­sion of lab­o­ra­tory tech­ni­cians, and we were rig­or­ously trained on the proper ex­e­cu­tion of the pro­to­cols used through­out the pro­ject.

Handling Chemicals #

We con­ducted bio­plas­tic syn­the­sis ex­per­i­ments un­der the con­stant su­per­vi­sion of our in­struc­tors. While work­ing with haz­ardous chem­i­cals such as acetic an­hy­dride, sul­fu­ric acid, hy­drochlo­ric acid, and ni­troben­zene, fume hoods and proper PPE were used.

Waste Disposal #

Sharps con­tain­ers and bio­haz­ard bins were main­tained at two sites in the lab, and the re­spec­tive wastes were care­fully dis­posed. Our Institute’s Safety cell es­tab­lished guide­lines, which were care­fully fol­lowed.

Experimental Safety #

Our trans­formed E. coli strains were Ampicillin and Kanamycin re­sis­tant due to the use of pCri and pET vec­tors re­spec­tively, whereas the trans­formed B. sub­tilis was re­sis­tant to Chloramphenicol (through pCri). Proper han­dling prac­tices and san­i­ti­za­tion pro­ce­dures were fol­lowed to avoid any leak­age into the en­vi­ron­ment.

Ethics #

As our pro­ject in­volved sur­vey­ing peo­ple and ex­tract­ing data from gov­ern­ment data­bases, we en­sured to fol­low all the eth­i­cal as­pects for col­lect­ing and han­dling this data. We fol­lowed the guide­lines in our re­gion by the gov­ern­ment and by iGEM for car­ry­ing out such ac­tiv­i­ties. Additionally, we con­sulted var­i­ous fac­ul­ties within our in­sti­tute and out­side for re­ceiv­ing guid­ance on car­ry­ing out sur­veys on hu­man sub­jects.

Image gallery #

References #

1. Sharifi R, Ryu C-M. Social net­work­ing in crop plants: Wired and wire­less cross-plant com­mu­ni­ca­tions.Plant Cell Environ. 2021;44:1095–1110. https://​doi.org/​10.1111/​pce.139661110SHAR­I­FIANDRYU
2. Baruah J, Nath BK, Sharma R, Kumar S, Deka RC, Baruah DC and Kalita E (2018) Recent Trends in the Pretreatment of Lignocellulosic Biomass for Value-Added Products.
3. Front. Energy Res. 6:141. doi: 10.3389/fenrg.2018.00141 Simeng Zhou, Sana Raouche, Sacha Grisel, Jean-Claude Sigoillot, Isabelle Gimbert. Efficient bio­mass pre­treat­ment us­ing the White-rot Fungus Polyporus Brumalis.. Fungal Genomics & Biology, Omics Publishing Group, 2017, 7 (1), pp.1-6. 10.4172/2165-8056.1000150.
4. Elbaz M, Ben-Yehuda S. 2015. Following the fate of bac­te­r­ial cells ex­pe­ri­enc­ing sud­den chro­mo­some loss. mBio 6(3):e00092-15. doi:10.1128/​mBio.00092-15. Brantl S, Müller P. Toxin⁻Antitoxin Systems in Bacillus sub­tilis. Toxins (Basel). 2019 May 9;11(5):262. doi: 10.3390/toxins11050262. PMID: 31075979; PMCID: PMC6562991.
5. iGEM12_U­N­AM_Ge­nomic­s_Mex­ico: http://​parts.igem.org/​Part:BBa_K851002
6. yqcG: http://​parts.igem.org/​Part:BBa_K3507002
7. eGFP: http://​parts.igem.org/​Part:BBa_K2075001