Pengklonan dan Pengekspresan Gen Xilanase Termostabil daripada Bacllus Coagulans St-6 ke dalam Escherichia Coli HB101

Bacillus coagulans ST-6 merupakan bakteria termofilik yang berupaya mengekspreskan aktiviti xilanase termostabil. Gen xilanase tersebut telah beIjaya diklonkan dengan menyelitkan serpihan DNA daripada genom B. coagu/ans ST-6 yang telah dipotong oleh enzim Bam HI ke dalam tapak Bam HI yang terdapat di dalam vektor pBR322. Seterusnya plasmid rekombinan tersebut ditransformasikan ke dalam Eshcerichia coli HB101 dan koloni yang mengandungi gen xilanase dipilih dengan menggunakan agar media bercampur substrat RBB-xilan. Daripada 3,270 transforman yang terpilih, hanya dua koloni didapati mengekspreskan aktiviti xilanase iaitu dengan kewujudan zon cerah di sekelilingnya. Penyaringan seterusnya mendapati hanya satu koloni sahaja yang stabil untuk kajian selanjutnya. Plasmid rekombinan tersebut dinamakan sebagai pBNX. Pemotongan plasmid pBNX dengan enzim Bam HI mendapati DNA selitan tersebut bersaiz 2.56 kb. Kajian selanjutnya menunjukkan ia mengandungi tapak pemotongan bagi enzim Hind 111, Sal 1 dan &0 RV tetapi tiada tapak bagi enzim Eco R1, Pst 1, Kpn 1 dan Sac 1. Keputusan daripada penghibridan dengan prob DNA selitan melalui kaedah pemblotan Southern telah mengesahkan DNA selitan bagi plasmid rekombinan (pBNX) tersebut berasal daripada B. coagu/ans ST-6. Enzim xilanase kasar yang dihasilkan oleh klon tersebut menunjukkan ciri-ciri yang sarna dengan enzim xilanase kasar daripada B. coagu/ans ST-6, di mana suhu dan pH optimumnya adalah SOoC dan 7.2. Enzim xilanase daripada E. coli (PBNX) dan B. coagulans ST-6 juga stabil pada suhu 60˚C. Pengsubklonan DNA selitan dalam plasmid pBNX ke dalam pUC19 dan pUC18 juga telah dilakukan dan kedua-duanya mengekspreskan aktiviti xilanase. Plasmid kedua-duanya dikenali sebagai pBNXl dan pBNX2. Tindakbalas dengan enzim pembatas Sal 1 menunjukkan cara kemasukan DNA selitan dalam kedua-dua subklon mempunyai orientasi yang sama

Protein expression of Late Elongated Hypocotyl (LHY) homolog genes of teak in Escherichia coli

Expression of an isolated gene in a system that directly translates it into a protein is an important step to study the protein encoded by the gene. The isolated gene can be expressed in vivo by a heterologous system. In this study, a bacteria system was used to translate the Tectona grandis Late Elongated Hypocotyl (Tg-LHY) gene, which was isolated from flowering tissues of teak (Tectona grandis). The gene was cloned into the pET 14b vector (Novagen) and transformed into BL 21(DE3)/pLysS and Rosetta 2 expression host cells (Novagen). Rosetta 2 host cell has been found to be a good candidate to express the Tg-LHY protein from plant origin, as it recognizes the codon that was found in plant but rarely used in bacteria. The expressed protein was about an expected size, which was 90 kD. Western blot analysis using antibody against His-tag, which was fused to the Tg-LHY protein, proved that the expressed protein was Tg-LHY protein

Compressive and Tensile Capacity of Recycled Aggregate Concrete (RAC) with Glass as Supplement Material

The amount of construction waste is increased significantly over the years due to reconstruction and the demolition of old buildings. One of the major challenges of our present society is to protect the environment by recycling the existing construction waste. This study concerned on two types of variable in the production of concrete which are the utilization of coarse recycled aggregate and utilization of different supplement ratio of fine glass wastes to cement. To evaluate the viability of this study, an experimental work was performed in order to monitor the mechanical behavior of such concrete. The compression and splitting tensile strength of concrete were determined on this study. From the result, it is conclude that the utilization of recycled aggregate does not much affect in the uniaxial compressive strength and splitting tensile strength of concrete, for replacement ratio up to 25 %. However, the utilization of fine glass as supplement material to cement is increase the uniaxial compressive and splitting tensile strength of concrete, for supplement ratio up to 5 %. Thus, it can be stated that the optimum concrete mixture is the mixture of 25 % recycled aggregate and 5% glass.&nbsp

Isolation and characterization of LHY homolog gene expressed in flowering tissues of Tectona grandis (teak)

Floral initiation of teak through molecular biology approach is being studied for better understanding of teak flower development. Through PCR subtractive hybridization method, LHY homolog gene has been isolated from teak flowering tissues. The full-length cDNA of the gene was 2948 base pair (bp) and potentially encoded for 768 amino acids. It was named Tectona grandis LHY (Tg-LHY), as the gene was similar to the LHY gene of some species. Amino acid sequence alignment revealed that Tg-LHY was similar to LHY of Castanea sativa, LHY ofPhaseolus vulgaris and LHY of Arabidopsis thaliana. The highly conserved region found in Tg-LHY was the MYB protein, which is the DNA-binding protein responsible in negative feedback loop reaction of central oscillator in plant circadian clock system. The level of gene expression was found to be high four hours after dawn in flowering shoots and flower. This paper reported the isolation and characterization of the gene

Analysis of expressed sequence tags derived from inflorescence shoot of Tectona grandis (teak)

Teak Inflorescence Shoot Stage 4 (TIS4) shoots bearing the floral meristems were used to construct a cDNA librariy with insert size range of 1500 - 5000 bp. The titer of the library was 7.5 x 105 pfu/ml (primary) and 4.5 x 109 pfu/ml (amplified). EST generation and analysis were performed using the cDNA library where a total of 1384 plaques were randomly picked and their inserts PCR-amplified using T3 and T7 universal primers. Only 1125 plaques generated single amplified fragments, each which were purified and sequenced using the SK universal primer. The generated raw 5’ ESTs were filtered and clustered. A total of 674 nonredundants (69 consensus sequences and 605 singletons) were generated and their identities searched through BLASTX. Of the 674 nonredundants, 107 of them (15.9%) showed no hits or no identity. All the 567 nonredundants identified through BLASTX were categorized into their functional categories and were further analysed using InterProScan to detect their protein signatures and to assign their GO numbers. From all the sequences analysed, only 186 (32.8%) sequences were given the GO numbers and grouped into the three GO main categories namely biological process, cellular component and molecular function. Several important ESTs were highlighted based on their functional categories. There were five sequences found to be related to flowering and light induction