Objective #

To come up with a syn­thetic bi­ol­ogy based so­lu­tion for degra­da­tion of stub­ble (crop residue left be­hind post har­vest) - so as to cir­cum­vent the prob­lem of its burn­ing.

Chassis #

To se­lect a chas­sis for our pro­ject, we used the fol­low­ing cri­te­ria:

1. The bac­te­ria should be non-path­o­genic to plants, hu­mans or an­i­mals.

2. The bac­te­ria should be able to with­stand high tem­per­a­tures and pH which may be preva­lent when in­tro­duced to agri­cul­tural fields.

3. Ease of cul­ti­va­tion in lab­o­ra­tory con­di­tions, which would help make ex­per­i­men­ta­tion eas­ier.

While fun­gal sys­tems are widely stud­ied for the break­down of lig­no­cel­lu­losic bio­mass, grow­ing fungi re­quires high strin­gency in con­di­tions, which makes it hard to trans­late our pro­ject to our field.

Hence, we ini­tially chose to work on Bacillus sub­tilis, a soil bac­terium which is re­sis­tant to harsh en­vi­ron­men­tal con­di­tions and can eas­ily be grown in the lab. However, upon dis­cus­sion with Mr. Malhar Atre (see Attributions), we learnt that Bacillus sub­tilis is a slightly dif­fi­cult sys­tem to work on - hence it would be bet­ter if we could work on Escherichia coli to demon­strate a proof of con­cept.

Hence, we de­cided to per­form the pro­tein ex­pres­sion stud­ies and func­tional analy­sis in the E. coli BL21 strain, post which we shall ex­trap­o­late our work to Bacillus sub­tilis.

Plasmid #

In line with this plan, we had to use a plas­mid which could be shut­tled be­tween E.coli and Bacillus sub­tilis. After much re­search, we ar­rived at pCri18-a - a mod­i­fied ver­sion of pH­T43 which has the fol­low­ing ad­van­tages:

1. Presence of a 6X His tag at the C-terminal, which en­ables easy en­zyme iso­la­tion.

2. Presence of a se­cre­tion sig­nal pep­tide (SamyQ - Biobrick BBa_K1074014) up­stream of the MCS en­sures ex­tra­cel­lu­lar stub­ble degra­da­tion.

3. Presence of a strong, well stud­ied pro­moter (Pgrac01 - Biobrick BBa_K1074012) which is IPTG in­ducible.

Constructs #

The plant cell wall, mainly com­posed of cel­lu­lose, pectin, xy­lan and lignin are the most re­cal­ci­trant parts of the stub­ble. Hence, we trans­formed bac­te­ria with genes which en­code en­zymes that de­grade each of the four com­po­nents of the cell wall thor­oughly.

IPTG in­duc­tion of the Pgrac pro­moter causes in­duc­tion of each of the four gene cas­cades, cloned into four dif­fer­ent colonies. The fol­low­ing de­grad­ing en­zymes are re­leased here:

1.Cellulases #

The cel­lu­lase con­struct is com­posed of two dif­fer­ent bio­bricks - a β glu­cosi­dase known as CglT(Biobrick BBa_K4382000) which helps break­down cel­lobiose to glu­cose. Another gene which is in­duced is the EG5C - 1 gene(Bio­brick BBa_K4382001), a pro­ces­sive en­doglu­canase which breaks down cel­lu­lose fib­rils into var­i­ous oligosac­cha­rides - mostly cel­lobiose and cel­lotriose.

2.Pectinases #

The pecti­nase con­struct is com­posed of two endo pec­tate lyases namely PelA(BBa_K4382007) and PelB-B2(BBa_K4382006) which help in break­ing down the pectin back­bone and a pectin methylesterase called Pme(BBa_K4382008), which helps cleave the methyl-es­ter branches of the galac­touronic acid chains of pectin.

3.Ligninases #

The lign­i­nase gene cas­cade is com­posed of BsDyP(BBa_K1336003) and DyP1B(BBa_K4382002); both of which are dye de­colouris­ing per­ox­i­dases which come to­gether to break­down lignin into its con­stituent monomers.

4.Xylanases #

The xy­lanase gene cas­cade is com­posed of XynA(BBa_K4382010) - an endo-1,4-β-xy­lanase that cleaves xy­lan back­bone to form xylo-oligosac­cha­rides , XynB(BBa_K4382003) - which cleaves xylo-oligosac­cha­rides to xy­lan monomers, XynC(BBa_K4382004) - which cleaves xy­lan into Methyl Glucuronoxylan units and XynD - which cleaves the xy­lo­sidic bond be­tween the xy­lan and the ara­bi­no­fu­ra­nose to re­lease ara­bi­no­fu­ra­nose(BBa_K4382005).

Planned work­flow #

The above pic­ture shows a schematic di­a­gram of the work­flow of our pro­ject. Post iden­ti­fi­ca­tion of the genes of in­ter­est and de­sign­ing the gene cas­cades, the next step is to lig­ate the genes into our plas­mid of in­ter­est and in­sert­ing it into Bacillus sub­tilis subsp. sub­tilis 168. Post this, pro­tein ex­pres­sion analy­sis fol­lowed by bio­chem­i­cal as­says to con­firm the func­tion­al­ity of each pro­tein.

All the ge­net­i­cally en­gi­neered bac­te­r­ial colonies shall be mixed and the for­mu­la­tion shall be ap­plied on the stub­ble. If this ap­pli­ca­tion of the for­mu­la­tion is done on the field, the de­com­posed stub­ble shall turn into a biofer­til­izer.

But if the spray­ing is done on stub­ble in-vitro, the vanillin thus pro­duced can be used to pro­duce bio­plas­tics.

Thus, we pro­pose a two pronged so­lu­tion to the prob­lem of stub­ble burn­ing - one is de­com­po­si­tion on the field and the other is de­com­po­si­tion of col­lected stub­ble to man­u­fac­ture bio­plas­tics.

Experimental Design #

To en­sure that our bac­te­ria get cleared off the field once the de­com­po­si­tion is achieved, we have the­o­ret­i­cally de­signed a kill switch in­volv­ing a pBAD-pXyl AND gate (BBa_K851002, sub­mit­ted by iGEM12_U­N­AM_Ge­nomic­s_Mex­ico) along with a down­stream GFP-tagged toxin gene yqcG (BBa_K3507002, sub­mit­ted by iGEM20_­Gronin­gen) in the cod­ing se­quence, and a dual stop codon as the ter­mi­na­tor. The pBAD-pXyl pro­mot­ers sense L-arabinose and D-xylose re­spec­tively, which are the di­ges­tion end-prod­ucts of ara­bi­noxy­lans, a com­par­a­tively slow-di­gest­ing ma­te­r­ial in stub­ble.

This has been elab­o­rated in greater de­tail in Safety.

Bioplastics #

Wheat Straws are treated with white liquor and neu­tral­ized with $H_{2}S{O}_4$ to get the lignin. On treat­ing lignin with NaOH and Nitrobenzene and fur­ther with $H_{2}S{O}_4$, we get the Vanillin us­ing Nitrobenzene Oxidation Method. Vanillin is re­fluxed in the pres­ence of Acetic Anhydride and Sodium Acetate to give Acetyl Ferulic Acid, which is col­lected over ice. Polymerization of Acetyl Ferulic Acid with $Zn(OAc{)}_2$ and melt­ing with glyc­erol will give us the bio­plas­tic mold, which can be molded into the de­sired shape.

Proof of Concept #

(+) in­ocu­lum and (–) in­ocu­lum were taken in the con­i­cal flask and in­cu­bated for 24 Hours. After Incubation, the Supernatant and Straw were fil­tered. Applying the lignin ex­trac­tion pro­ce­dure (From the Bioplastics Experiment) and test­ing the pres­ence of lignin through Safranin Test. In the su­per­natant, the pres­ence of lignin was tested through the safranin test and con­firmed the lignin degra­da­tion in the su­per­natant through the 2,4 — DNP test.

References #

1. Goulas, T., Cuppari, A., Garcia-Castellanos, R., Snipas, S., Glockshuber, R., Arolas, J. L., & Gomis-Rüth, F. X. (2014, November 11). The pCri System: A Vector Collection for Recombinant Protein Expression and Purification. PLoS ONE, 9(11), e112643. The pCri System: A Vector Collection for Recombinant Protein Expression and Purification | PLOS ONE

2. How to han­dle Bacillus sub­tilis - https://​sta­tic.igem.org/​me­di­awiki/​2020/​5/​50/​T–Brno_Czech_Re­pub­lic–Con­tri­bu­tion_Hand­book.pdf