Experimental flow­chart to de­scribe the proof of con­cept #

(+) in­ocu­lum (XynC (BBa_K4382004) and BsDyP (BBa_K1336003)) 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 was through the 2,4 — DNP test.

Results #

ACTIVITY TEST OF ENGINEERED BACTERIA: Lignin is one of the most re­cal­ci­trant ma­te­ri­als pre­sent in the plant cell walls, which is very dif­fi­cult to de­grade. Lignin in­ter­links with other car­bo­hy­drates like cel­lu­lose, hemi­cel­lu­lose, and xy­lans to form a strong mesh. We wanted to test the ac­tiv­ity of our en­gi­neered bac­te­ria with en­hanced ex­pres­sion of the lign­i­nase and xy­lanase genes in vitro. To in­ves­ti­gate this, we treated chopped wheat straw with our en­gi­neered bac­te­r­ial cul­tures.

Figure 1: (a) Safranin test to de­tect lignin in the con­trol sus­pen­sion with­out bac­te­r­ial in­ocu­lums gave pink­ish-red color, whereas the test (with bac­te­r­ial in­ocu­lums) did not show any sig­nif­i­cant color change. (b) The com­pound lignin ex­tracted by chem­i­cal treat­ment of straw cor­re­sponds to con­trol. (c) A com­pound ex­tracted by chem­i­cal treat­ment of straw cor­re­spond­ing to bac­te­r­ial in­ocu­lum treat­ment.

We used the clones ob­tained, XynC (BBa_K4382004) and BsDyP (BBa_K1336003), to treat the wheat straw. After treat­ment for 24 hours, we pro­ceeded with a safranin test to de­tect the pres­ence of in­tact lignin. We ob­served a change in the colour of our con­trol bac­te­r­ial sus­pen­sion as com­pared to that of bac­te­r­ial in­ocu­lum-treated straw (test). This in­di­cates the ab­sence of lignin in the test sus­pen­sion, which might be pos­si­bly due to the ac­tion of BsDyP ((BBa_K1336003)). We also ob­served a change in lignin mass from the treated straw (b and c). The bac­te­r­ial in­ocu­lum-treated straw ex­trac­tion mass is 0.67125 gm from 1gm of straw, whereas the con­trol straw could yield 0.832gm lignin. This in­di­cates the lignin con­tent in treated straw is more de­graded than the con­trol.

Figure 2: (a) In the Safranin test, lignin was de­tected for the con­trol straws (left) but it was not de­tected for the test straw (middle). Pure Safranin (right) was taken as an ex­per­i­men­tal con­trol.
(b) The bac­te­r­ial cul­ture cor­re­spond­ing to the test gives a pos­i­tive test by giv­ing an or­ange colour with 2,4 DNP, in­di­cat­ing that the lignin is de­graded, whereas no colour change is ob­served in the con­trol, in­di­cat­ing that the lignin is not de­graded.

We fur­ther pro­ceeded with the straw for the lignin de­tec­tion test. We did not ob­serve any change in lignin con­tent in the con­trol and test. Since the lignin con­tent var­ied in the su­per­natant ob­tained af­ter de­com­po­si­tion, we pro­ceeded with the 2,4-DNP test to de­tect the pres­ence of aro­matic com­pounds. the test ex­tract showed a change in color, in­di­cat­ing the pres­ence of aro­matic com­pounds. This es­tab­lishes the fact that our straw treated with BsDyP ex­pres­sion con­struct is able to de­grade lignin and for­ward the re­ac­tion to the for­ma­tion of aro­matic com­pounds.

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