In planta expression of hyperthermophilic enzymes as a strategy for accelerated lignocellulosic digestion


S1: Prediction of cleavage and N-glycosylation sites of recombinant endoglucanase (a) and recombinant xylanase (b). Sub-cellular localization of the recombinant proteins was predicted using TargetP (Emanuelsson et al., 2000(Emanuelsson et al., , 2007) ) and N-glycosylation sites were predicted using NetNGlyc (http://www.cbs.dtu.dk/services/NetNGlyc/). a S2: Gene cloning of Rec-EG (2756bp), and Rec-Xyn (1146bp) using gene specific primers. M= marker ladders. PCR for EG gene was performed with the following conditions: 30s at 98°C; 30 cycles as follows: 10 s at 98°C; 40 s at 60°C; 1 min at 72°C; 5 min at 72°C. PCR for Xyn was performed with the following conditions: 30s at 98°C; 30 cycles as follows: 10 s at 98°C; 30 s at 60°C; 40s at 72°C; 5 min at 72°C. 

bS3:

  Endoglucanase and xylanase activity of protein extracts from C108 and X7 plants expressing Rec-ebi244Opt and Rec-rMxylOpt respectively. A) Observation of clear zone around the protein extracts on 0.1% RBB-xylan (Sigma) plate using 0.1% Congo Red. B-C) Observation of clear zone around the protein extracts on 1% CMC (Sigma) plates using 0.1% Congo Red. The plates were incubated at 75°C for 30 min. 




  




  


Table 1 : Primers used in this work

 1 
	Primer name	Sequence (5'-3')
	EGFW1	CAGCTGGCCACCATGGGATTTGTTCTCTT
	EGRev1	CAGCTGTTAGCCAGCCCTAGTGATTTCG
	XynFW1	CAGCTGGCCACCATGGGATTTGTTC
	XynRev1	CAGCTGTTACGGCGTGTTTCCGTAG
	EGFW2	CAGCTGGCCACCATGGGATTTGTT
	EGRev2	CAGCTGTTAGCCAGCCCTAGTGAT
	XynFW2	CAGCTGGCCACCATGGGATTTGTT
	XynRev2	CAGCTGTTACGGCGTGTTTCCGTA
	EGFW3	CAGCTGGCCACCATGGGATTCGTG
	EGRev3	CAGCTGTTATCCTGCCCTAGTAAT
	XynFW3	CAGCTGGCCACCATGGGTTTCGT
	XynRev3	CAGCTGTTAAGGTGTATTACCATATCC
	EGFW4	TGTCAGAAATTAGAGAGTTTATGGTTA
	EGRev4	TAATTGTAGTGGTAGGTGTCTGAGGTG
	EGFW5	GATACGGACCAGAAGGTTTTAAGGATA
	XynFW4	CTTCATCAAGTTCTTCAAGTTCTGGAG
	XynRev3	AAAGGGTTTGTAGTCCATCCATACAAT
	RT-PCR/qRT-PCR-Xyn-FW	CAGACCTCCAGGAGGTCAAGGGGTCCAGTT
	RT-PCR/qRT-PCR-Xyn-Rev	AGTACCTCCGGTCCTTCTCTGTTCGGCATGC
	RT-PCR/qRT-PCR-EG-FW	GAATAGGATTGGTTCCATGGGCTCCGTCGGA
	RT-PCR/qRT-PCR-EG-Rev	TATCTCCCCAATTATCTGCGAGCGTTGCGA
Bilal Ahmad Mir, Alexander A Myburg, Eshchar Mizrachi, Don A Cowan
In planta expression of hyperthermophilic enzymes as a strategy for accelerated lignocellulosic digestion