Acclimatization of transformed seaweed Kappaphycus alvarezii carrying lysozyme gene in culture flask and the cultivation in floating net cage in Pangkep coastal

The in vitro transformation of the lysozyme gene in seaweed K. alvarezii has been successfully executed to increase the viability against ice-ice disease. There were two major stages in this research; (1) transformation of lysozyme gene in seaweed K. alvarezii which was carried out on laboratory scale and the cultivation of gene-transformed explants in the culture flask stored in “culture chamber”; (2) the acclimatization in floating net cages of green nets (mesh size of 1 mm) with cage size of 50 x 50 x 50 cm, the population density of 200 explants and cultivated for two weeks. The explants were then transferred to blue nets (mesh size of 2 mm) with a cage size of 50 x 50 x 50 cm for four weeks of rearing. The plants were then enlarged using a long-line method in the floating net cage, by tying the seaweed using a double line with a gap of 15 cm each. The measurement of weight, bud lengths, and water quality was carried out within 2 weeks. The result shows that the daily growth rate of the transformed seaweed during the regeneration stage in the culture flask was around 0.33-0.4%/day, meanwhile during the acclimatization stage in the green nets the was 0.65-1.6%/day, and even more, increased during the acclimatization stage in the blue nets with DGR of 2.28-2.3%/day. During the enlargement stage in the floating net cages, the lysozyme-transformed seaweed showed an even higher DGR with a value of 3.2-8.2%/day. The results of the integration of the lysozyme gene in seaweed were indicated by the presence of a 670 bp of amplification products, that is the same total length of the 35 S-F promoter fragments and Nos T-R in the expression vector. Based on these results, the lysozyme gene was successfully transformed in K. alvarezii seaweed.

Determination of volatile organic compounds exhaled by cell lines derived from hematological malignancies

Background: The gas human exhaled contains many volatile organic compounds (VOCs), which is related to the health status of body. Analysis of VOCs has been proposed as a noninvasive diagnostic tool for certain cancers. Detailed research on the VOCs in gas exhaled by cell can characterize cell type specific metabolites and may be helpful to detect the cancer markers in clinical practice. Methods: Solid-phase microextraction (SPME) gas chromatography–mass spectrometry was used to detect VOCs in the headspace of tissue culture flask in non-Hodgkin’s lymphoma (NHL) cell line JEKO and acute mononuclear leukemia cell line SHI-1, to elaborate the characteristic gaseous biomarkers of hematological malignancies. While macrophage cells and lymphocytic cells were acted as control. The blank group was only the RPMI 1640 medium containing 10% fetal calf serum that without cells. Results: Comparing with control group, the concentration of dimethyl sulfide, 2,4-dimethylheptane, methylbenzene, o-xylene, dodecane, and 1,3-di-tert-butylbenzene in JEKO cells was relatively higher, while the concentration of ethanol, hexanal, and benzaldehyde was lower. In SHI-1 cells, the levels of 2,4-dimethylheptane, benzene, 4-methyldecane, chloroform, 3,7-dimethyl dodecane, and hexadecane were significantly elevated, but the levels of hexanol and cyclohexanol were distinctly reduced. Conclusions: This pilot study revealed that the malignant hematological cells could change the components of VOCs in the cell culture flask in a cell type-specific pattern. The traits of VOCs in our setting offered new strategy for hematological malignancies tracing, and would act as potential biomarkers in diagnosis of malignant hematological diseases.

REGENERASI DAN PERBANYAKAN RUMPUT LAUT Kappaphycus alvarezii HASIL TRANSFORMASI GEN SUPEROKSIDA DISMUTASE (MaSOD)

Transformasi gen superoxide dismutase (MaSOD) pada rumput laut Kappaphycus alvarezii menggunakan Agrobacterium tumefacient telah dilakukan secara in vitro. Transformasi gen MaSOD ke dalam genom rumput laut diharapkan dapat mengurangi cekaman oksidatif terutama yang disebabkan oleh perubahan suhu, salinitas, dan cemaran logam di perairan. Penelitian ini bertujuan untuk regenerasi rumput laut hasil introduksi gen MaSOD dan non-transgenik pada labu kultur. Regenerasi dan perbanyakan rumput laut hasil transformasi gen MaSOD dilakukan di laboratorium pada labu kultur yang diletakkan dalam “culture chamber” yang dilengkapi dengan aerasi menggunakan media kultur yang diperkaya dengan pupuk PES, Grund, Conwy, dan SSW sebagai kontrol, salinitas 20, 25, 30, 35, dan 40 g/L, pH 4, 5, 6, 7, dan 8. Intensitas cahaya antara 500-2.000 lux dengan fotoperiode terang dan gelap 8:16; 12:12; dan 16:8. Untuk merangsang pertumbuhan eksplan dilakukan pemeliharaan dengan penambahan hormon tumbuh IAA dan BAP dengan perbandingan 1:1, 1:2, dan 2:1. Penelitian dilakukan secara bertahap. Evaluasi transgenik dilakukan menggunakan teknik PCR. Hasil penelitian memperlihatkan bahwa sintasan yang paling tinggi diperoleh menggunakan media PES (94%), salinitas 30 g/L (90%), pH 7 (96%), intensitas cahaya pada 1.500 lux (80%), fotoperiode 12:12 (84%), komposisi ZPT dengan campuran IAA dan BAP dengan perbandingan 2:1. Hasil analisis PCR memperlihatkan K. alvarezii transgenik putatif mengandung transgen MaSOD sebanyak 78% dari hasil transformasi.Superoxide dismutase transformation (MaSOD) gene of seaweed Kappaphycus alvarezii mediated by Agrobacterium tumefacient has been successfully done in vitro. MaSOD genes introduced into the seaweed genome is expected to reduce oxidative stress caused by environmental conditions such as changes in temperature, salinity and metal contamination of the water. This study aimed to regenerate both the MaSOD transformed seaweed and non-transgenic in a culture flask. Regeneration and multiplication of those seaweed were conducted in a laboratory flask cultures placed in a “culture chamber” which was aerated and enriched with fertilizers PES, Grund, Conwy, and SSW as a control, salinity 20, 25, 30, 35, and 40 g/L, pH: 4, 5, 6, 7, and 8. the light intensity between 500-2000 lux, with light and dark photoperiod 8:16; 12:12; and 16:8. To stimulate the growth of explants the addition of growth hormone IAA and BAP with ratios of 1:1, 1:2 and 2:1 were performed. This study was a multiple-step of process, by which transgenic explants was identified by PCR method. The results showed that the highest survival rate was obtained using media PES (94%), salinity 30 g / L (90%), pH= 7 (96%), the intensity of light at 1500 lux (80%), photoperiod= 12: 12 (84%), and ratio of IAA and BAP 2: 1. The results of PCR analysis showed the putative K. alvarezii transgenic MaSOD was 78% of explants.

Screening of Biosurfactant-Producing Bacteria

Firstly, two biosurfactant-producing bacteria named 1# and 2# was selected from 7 strains. Secondly, their growth and metabolism laws that the quantity of metabolic products would reach the highest and the value of surface tension would bottom out in the stationary phase of growth was found in the culture flask. Thirdly, the optimal condition for fermenting was chose by the orthogonal test which was used to study the effect of constituent of fermentation medium, culturing temperature and rotation speed of shaking table on the capability of metabolizing biosurfactants. Finally, compared the interfacial tension of strain 1# with that of strain 2# at the optimum conditions, strains 1# was chose for industrial fermentation.