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Home > The Role of the Ribosome Binding Site Sequence and Spacer Length Between Binding Site and Initiation Codon on Cry2 Expression in Bacillus Host

The Role of the Ribosome Binding Site Sequence and Spacer Length Between Binding Site and Initiation Codon on Cry2 Expression in Bacillus Host

Thesis Info

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External Link

Author

Rooma Adalat

Program

PhD

Institute

Lahore College for Women University

City

Lahore

Province

Punjab

Country

Pakistan

Thesis Completing Year

2018

Thesis Completion Status

Completed

Subject

Biotechnology

Language

English

Link

http://prr.hec.gov.pk/jspui/bitstream/123456789/10793/1/Rooma%20Adalat_Biotech_2018_LCWU_PRR.pdf

Added

2021-02-17 19:49:13

Modified

2024-03-24 20:25:49

ARI ID

1676727596556

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The bacterium Bacillus thuringiensis (Bt) produces Cry toxins that possess toxic properties and can be used as biopesticides. Cry2Aa and Cry2Ac are among unusual subset of crystalline proteins possessing broad insect species specificity by exhibiting high specific activity against larvae from two insect orders, Lepidoptera and Diptera of agricultural and public health significance. The cry2Ac11 gene is located at third position (orf3) in operon comprising of three genes. It needs accessory proteins for crystal formation and high yield. Translation initiation is key rate-limiting step. It is well-known that stable structure at a ribosome binding site (RBS) impedes initiation. Modification in RBS-spacer region tunes translation initiation rates. Genetic manipulation of cry2Ac11 gene without helper protein was carried out in this study by optimizing ribosomal binding site and spacer region (RBS-ATG) in translation initiation region (TIR). The five different types of mutations were introduced in TIR to unveil inhibitory and excitatory effects on translation. These mutants are: 1), operSalI/RBSD and mut/RBSD in which downstream RBS (GGAGG) 6 bp downstream to native RBS was introduced in TIR of cry2Ac11 operon and gene; 2), mut/RBSF in which four nucleotides (ATGGG) were incorporated after RBS-ATG spacer region; 3), mut/RBSSin which overlapping start and two stop codons were introduced after RBS-ATG spacer region; 4), mut/RBSSP in which RBS-ATG spacerregion waslengthened to 23 nucleotides; 5), mut/RBS2 in which consecutive two ATGs were incorporated in TIR. Secondary structures of mutants, estimated by CLC Main Workbench, revealed that mut/RBS2 RNA exhibits most stable structure in RBS-AUG region. RBS Calculator predicts high translation rate in mut/RBSD and mut/RBS2. Mutants were expressed in B. thuringiensis 4Q7 acrystalliferous strain. The transcriptomics-proteomics profiles of all cry2Ac11 constructs provide a unique opportunity to investigate how faithfully the transcriptional profile is manifested at the protein level. Therefore, in this study correlation between mRNA abundance and protein expression profiles in all Cry2Ac11 recombinant strains were also investigated. The highest transcript profile of B. thuringiensis 4Q7-mut/RBS2, (a mutant in which consecutive multiple AUG were introduced), was obtained by Real time PCR. Furthermore, SDS-PAGE profile of total cellular proteins indicated that overexpression of Cry2Ac11 (65kDa) was obtained in 4Q7-mut/RBS2. It was concluded that overexpression of Cry2Ac11 toxin without helper protein in mut/RBS2 mRNA was most likely due to presence of consecutive start codons (AUGs) in TIR. Presence of RBS in the single stranded part of moderately stable hairpin loop (ΔG = 8.7 kcal/mol) in mut/RBS2 facilitates the interaction of RBS to the complementary 16S rRNA sequences of 30S ribosomal subunit. In proposed model, multiple factors are thought to contribute in translation efficiency of mut/RBS2 (cry2Ac11 mutants without helper protein) which includes stabilizer sequence at 5′ and 3′ ends, the availability of the RBS for binding to the anti-SD of 16S rRNA of 30S ribosomal unit and optimal context of RBS-AUG region provided by multiple AUGs in TIR.
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جیدی یاد توں جان لکائی ہوئی اے

جیہدی یاد توں جان لکائی ہوئی اے
اوہو یاد ہر ویلے ای آئی ہوئی اے
دکھ گھل دے سارے ول ساڈے
پنڈ دکھاں دی مہُرے ای چائی ہوئی اے
دکھ درد دا نہیں احساس کردے
یاری بے درداں نال لائی ہوئی اے
جان جندڑی روح تے دوس کوئی نہیں
ہر شے ای ایتھے پرائی ہوئی اے
تیری یاد وچ وقت جو گزر جاوے
اٹھے پہر دی ایہو کمائی ہوئی اے
چور چوہدری جگ نوں مت دیندے
عقل مند دی جگ ہنسائی ہوئی اے
موڑ پرت مہار ہن ول ساڈے
سنیا ہے بہار وی آئی ہوئی اے

Development of Micro, Small and Medium Enterprises (Msmes) Based on Creative Economy (Ekraf) and Gig Economy in Gorontalo Regency

The purpose of this study is to determine the development of MSMEs in Gorontalo Regency and to provide new innovations in the development of MSMEs based on Creative Economy (Ekraf) and Gig economy in Gorontalo Regency. This study uses a descriptive qualitative approach, which describes and describes facts according to the phenomenon of how development is seen from the strengths, weaknesses, threats and opportunities (SWOT analysis) with the aim of providing an Creative Economy-based MSME development strategy and the Gig economy in Gorontalo Regency. Data collection was carried out through observation, interviews and documentation about the strengths, weaknesses, opportunities, and threats of MSMEs against informants from three business fields, namely the Food Business Sector (dodol processed food), clothing (Sulaman Karawo) and handicrafts spread across three existing sub-districts in Gorontalo Regency. The results showed that the creative economy-based and gig economy-based MSMEs in Gorontalo Regency have not been fully implemented by MSME actors because they have limited capabilities and experience problems in their business development, this is due to several problems faced such as limited capital, raw materials and the use of simple production equipment, limited marketing media due to the absence of a MSME center, the types of products offered, quite a lot of transaction costs, less skilled labor, and product copyright registration problems for that from the results of the SWOT analysis of the diversification strategy at a more precise value to be applied, because it is considered more appropriate to the current conditions in Gorontalo Regency. In addition, the use of the gig economy is a part-time worker who is competent and innovative for MSMEs that are progressing and ready to compete.

Taxonomic Studies of Two Major Genera Polygonum L. Complex and Rumex L. of Polygonaceae from Pakistan

The detailed taxonomic study of the Complex genus Polygonum L. (segregated into six genera and discussed separately in the present account) and Rumex L. of Polygonaceae has been carried during 2006-2009. Material for morphological, anatomical and palynological studies was obtained from the herbarium specimens and freshly collected material while for Amplified Fragment Length Polymorphism (AFLP) only fresh samples were used. Forty eight species belonging to seven genera, Aconogonon (Meisn.) Reichenb., Bistorta Adans., Fagopyrum Mill., Fallopia Adans., Persicaria Mill., Polygonum L. and Rumex L. were morphologically reviewed in detail, original observations being supplemented by and compared with previously published information. 6 new taxa were described first time in this account and proposed to be the new varieties. These included three varieties of Polygonum aviculare L., two varieties of Polygonum paronychioides C. A. Mey. ex Hohen and one variety of Polygonum polycnemoides Jaubert & Spach. Leaf epidermal studies have been carried out on forty seven species of the family and observations are made through traditional light microscopy. To my knowledge no such work is yet known which has dealt with comprehensive qualitative and quantitative micromorphological foliar investigations in the taxa of Polygonaceae from Pakistan. The study revealed some distinctive anatomical features on adaxial and abaxial leaf surfaces such as epidermal cells outline, single or mixed type of stomata on the single leaf surface and presence or absence of trichomes. The shape of epidermal cells is found to be variable; six different stomatal patterns have been examined, including cyclocytic, pericytic and staurocytic stomata not reported earlier for the family. Variation among glandular and non glandular trichomes is also examined. Glandular trichomes are of peltate, capitate and spheroidal type while five types of non glandular trichomes are also examined. Crystalliferous cells have been reported first time in Rumex nepalensis Spreng. Although analyzed epidermal characters have systematic value but they are likely to be stable at specific and generic level and are not much helpful in generic delimitation.Pollen morphological characters have been investigated by light microscope (LM) and scanning electron microscope (SEM). Both qualitative and quantitative characters including aperture number, pollen shape, size in polar and equatorial view, colpi length, exine and intine thickness and exine sculpturing in both LM and SEM have been observed. The characters employed, have been proved to be useful in classification of the complex genus Polygonum L. into different genera. On the basis of aperture number and exine sculpturing pattern in SEM, 14 main pollen types and 6 subtypes namely, Aconogonon type, Bistorta type, Fagopyrum type, Dumetorum type, Convolvulus type, Persicaria type (further subdivided into three subtypes), Capitata type (subdivided into three subtypes), Plebijum type, Patulum type, Cognatum type, Avicularia type, Chalepensis type, Acetosa and Dentatus type have been identified. These different pollen types are arranged in three parallel directions to establish evolutionary trends in pollen types of seven genera of the family Polygonaceae. To examine inter and intraspecific relationship among 28 accessions of 13 species belonging to four genera, AFLP (Amplified Fragment Length Polymorphism) marker system was first time used. Single primer pair was used to amplify AFLPs and fragments were separated in 6% denaturing acrylamide gels. A total of 131 fragments were analyzed. According to present results, the AFLP knowledge was found to be sufficiently susceptible to identify small level of variations and can differentiate highly interrelated genotypes.