Molecular Biotechnology

Table of Contents

Introduction.

Task 1.

Task 2.

Task 3.

Task 4.

Conclusion.

References.

Introduction to Molecular Biotechnology

Experimental design of plasmids helps in the understanding and application of genetic tools that are invariably important to our existence. All the natural organisms are born with specific immunity which is rare and unique to it's genomics, proteomics and metabol-omics. The genetic and technological manipulation provides a basis for transformation and if the transformation is successful,then there is commercialization of the product. Just like humulin developed by Eli Lilly to produce human insulin in E.Coli for mass production through bioreactor. The main objective of the experiment is to produce temperature and salinity resistance experimental design for ectoine expression in Synechococcus elongatus.

Molecular Biotechnology - Task 1

Overall diagrammatic representation of plasmid construction starting from inserting of ect ABC to the final plasmid construct

Fig.1 - Overall diagrammatic representation of plasmid construction starting from inserting of ect ABC to the final plasmid construct.

(Source: Author’s creation)

The plasmid construct is important to define how the foreign gene will be inserted and how it will function depending on its insertion into the plasmid. The plasmids used should have a high copy number and the product introduced should not cause intentional inactivation. This is an implied example of plasmid construct which is stunned up and transformed into the Synechococcus elongatus PCC 7942,a model organism. With high throughput technology the final plasmid is made. The subcloning is done to gain functionality and to study the desired gene in more detailed information and description. The figure represents how the plasmid vector is constructed. It is a small demonstration of theoretical framework in biotechnology.

Molecular Biotechnology - Task 2

1.Sequencing, analyzing ectoine gene cassette of the extremophilic bacteria Halomonas elongata from www.ncbi.nlm.nih.gov by using forward and reverse primers. The whole cassette of gene ectoine is selected.

2. The cassette of the gene is incorporated into pCR2.1 vector (which has f1 and pUC1 ori with T's site for cloning)-Invitrogen™

3.Vector system-pCR2.1 +entABC plasmid is designed

4. Sub Cloning is done with pSK+IBAT(so that functionality of ent ABC is shown under Pet.

5. Transformation is performed in Synechococcus elongate PCC-7942 se1Bb1s-eYFP followed by transformation procedure of Golden

Golden & Steams,1988)

6. Bioreactor cultivation (Growth and ectoine synthesis in recombinant S.elongatus)

7.Analysis-(Ono et al., 1999)

  • Biomass of the measured spectrometrically at 700nm and concentration of glucose is measured by HPLC
  • Ectoine concentration is measured by HPLC (amino NH2 column)with an isocratic gradient of the mobile phase.
  • Protein detection is done by Bradford using bovine serum albumin as standard
  • UV detection at 225nm for amino acids
  • Autoradiography

8.Protein Separation and identification Ectoine from the transformed cells

  • Ectoine transformants are separated out by Disc replication by using microbiological procedures and growing
  • SDS-PAGE (Gel electrophoresis)
  • Mass Spectrometric Protein Identification
  • Band of interest is cut off
  • Samples analyzed by (AP) MALDI/TRAP-XCT mass spectrometer in-MS mode(Cánovas et al., 1997)
  • Assay of Ect A proteins by acylation and coupled Ect B and Ect C.

Molecular Biotechnology - Task 3

Sequence Analysis of Ectoine Gene from H. Elongata

Experimental design- 3532bp fragment from the Halomonas was obtained with the help of its conserved sequences by performing SEFA PCR (SElf-Formed Adaptor PCR) and 2423bp entABC was selected along with 980bp sequence upstream and 129 sequences downstream and were cloned from Halomonas sp. with Genbank accession no: AJ011103. The sequences were identified by using reverse and forward primers. Ent a is around 579 bp, ent b is 1269bp and ent c is around 390bp. These genes are under the same transcription unit producing 21.2kDa, 49.4kd, and 14.7kd and these were the control of d0 and d1 promoters(Rouches & Lambert, 2020).

Vector System and Subcloning

Experimental design- the vector pCR2.1 and the cloned sequence from Halomonas were inserted together then it was also subcloned with pSAK-IBAT which were experimentally designed by a group of scientists() and the desired site was introduced very carefully under Pet strong promoters and ent ABC has Xha1 and BamHI restriction endonuclease sites. So, the insert was under two origins of replication and three genes have the same transcriptional unit. The selectable markers used were ampicillin resistance and tetracycline resistance. The subcloning was done to get a good amount of ectoine protein depending on the regulation by Pet. The clone site of pCR2.1 was a TA site, initially, the ectoine gene was introduced there with pUC origin and PLac operon system (Zhang et al., 2020).

Transformation in Synechococcus Elongatus -PCC 7942

Experimental design- the Synechococcus were transformed with plasmid PASK-IBAT of 5200bp and were transformed according to Golden, 1987. The bacterium was grown in medium of (concentrations in mg liter1): (NH4)2SO4(6,000), K2HPO4(4,400);KH2PO4(3,400), CaCl26H2O (90), MgCl26H2O (1,100), glucose (10,000),ZnCl (3.5), MnCl2(0.46), CuCl2(7) and other sources of iron and molybdenum. And the transforming cells were selected by the presence of eYFP which acts as the selectable marker.

Bioreactor Cultivation, Analysis, and Protein Sequencing Were Done by Using the Known Protocols

Molecular Biotechnology - Task 4

Understanding the concept of ectoine pathway- Extremophlic bacteria like Halomonas elongata is an excellent example of osmoregular organism.it has cassette of gene which helps the organism to survey in the euryhaline condition of salt marshes and other brackish regions. These concepts intrigued the scientists and that started implying this technology in the plant biotechnology to make GMO crops especially in the crops that grow in extreme saline conditions such as the drought crops. The ectoine gene is a cluster of genes under the etc ABC and has a single transcription unit. Ent a codes for acetyl transferase, Ent b codes for amino transferase, and ent c codes for ectoine synthase. These are inserted under Ptet promoter.in our experiment and then it is introduced into Synechococcus elongatus. These bacterium is model transforming bacteria which was used by Golden in 1988 to simulate the utilization of the glucose in the bacteria.with the advancement and technological innovation there are more model species where this kind of experiments are performed to genetically modify our our crops to need the meeting demands of a growing population and it also helps in the protection and reviving of species in extreme conditions which has stenohaline tolerance. The methods and materials are explained as per the model species and its efficiency to transform and give higher amounts of ectoine which helps the organism to survive in various degrees of salinity. With these approaches we can also solve the problems of rising salinity in our soil with the increase in global warming.

Materials:

  • https://www.ncbi.nlm.nih.gov/nuccore/AJ011103.2?report=graph&rid=EUV8XN55016[AJ011103.2]&tracks=[key:sequence_track,name:Sequence,display_name:Sequence,id:STD1,category:Sequence,annots:Sequence,ShowLabel:true][key:gene_model_track,CDSProductFeats:false][key:alignment_track,name:other%20alignments,annots:NG%20Alignments|Refseq%20Alignments|Gnomon%20Alignments|Unnamed, shown:false]&v=0:3012&appname=ncbiblast&link_loc=fromHSP
  • Bacterial growth conditions 
  • Vectors-pCR2.10 with pUC ori with Ampicillin resistance and Kanamycin resistance.
  • Sub cloning vectors-Pask-(3246bp) with tetracycline resistance and ampicillin resistance and has F1 origin and pUC origin
  • Synechococcuselongatus PCC 7942(for transformation and vector expression)Se1Bb1s-eYFP
  • PCR machine and all the materials for running the PCR including PCR reagents from Invitrogen ™.
  • Selectable markers will be represented by eYFP which is combined with transforming vectors.
  • Autobiography machine to see the change in the DNA of the transformed cell.
  • Gel electrophoresis and all reagents to run Protein SDS gel electrophoresisIdeal option for PhastGel™ users(Ready-to-use gels at 10% and 15% with 250 x 125 x 0.45 mm, pre-formed slots)[HPE™ BlueHorizon™ / Multiphor II™ compatible] - To quantify the amount of ectoine produced.

Methods:

  • Sequence analysis of the ectoine gene from H.elongata- the material is found in ncbi.pubmed as the H.elongata Genbank accession number was provided. The reserve and forward primers were based on the basis of the entABC conserved region -5-ATG ACG CCT ACA ACC GAG AAC TTC A-3and 5-TCA ATC GAC CGG TGC GTA-3 from the database ncbi.gov (Sengupta et al., 2020)
  • Vector system and subcloning- After this vector system are designed as per task 1-the vector pCR2.1 and the cloned sequence from Halomonas were inserted together then it was also subcloned with pSAK-IBAT which were experimentally designed by a group of scientists() and the desired site was introduced very carefully under Pet strong promoters and ent ABC has Xha1 and BamHI restriction endonuclease sites. So, the insert was under two origins of replication and three genes have the same transcriptional unit. The selectable markers used were ampicillin resistance and tetracycline resistance. The subcloning was done to get a good amount of ectoine protein depending on the regulation by Pet. The clone site of pCR2.1 was a TA site, initially, the ectoine gene was introduced there with pUC origin and Plac operon system.
  • Transformation in Synechococcuselongatus-PCC 7942- the organism is grown in proper nutrition medium containing 5% v/v of Carbon sources like glucose, iron, and molybdenum with inducer gene IPTG and reporter gene eYFP with LacI gene promoter which increases the production of eYFP 24 fold times higher. There have been no significant synthetic systems of gene expression for controllable genes in Synechococcuselongatus PCC-7942. The culture was done at 30°C with 100 mL glass bottles under uniform and continuous fluorescent light (100 μmol photons/m2/s) in BG-11 medium (1.5 g/L NaNO3, 0.006 g/L ferric ammonium citrate, 0.001 g/L Na2EDTA·2H2O, 0.039 g/L K2PO4, 0.075 g/L MgSO4·7H2O, 0.020 g/L Na2CO3, 0.036 g/L CaCl2·2H2O, 0.006 g/L citric acid, 2.860 mg/L H3BO3, 1.810 mg/L MnCl2·4H2O, 0.222 mg/L ZnSO4, 0.39 mg/L Na2MoO4·2H2O, 0.079 mg/L CuSO4·5H2O, 0.0494 mg/L Co[NO3]2·6H2O) supplemented with 10 mM MOPS (pH 7.5)(Hirokawa et al., 2020)The culture was supplied with a constant rate of 10 mL/min into the medium was 5% (v/v) CO2 gas and 95% (v/v) air (Sato et al., 2007). The selection process was with 10 μg/mL ampicillin 10 μg/mL and kanamycin. The inducer Isopropyl-β-D-1-thiogalactopyranoside (IPTG) was added to the medium which was inoculated for the induction process. The growth was evaluated by OD730 with a UV spectrophotometer which shows the recombinant cells. The BG-11 medium plus the reporter gene was kept overnight at 30°C to induce the expression of eYFP and fluorescence intensity test was performed using a kit (Tecan Infinite M200 Pro, Tecan Group Ltd., Switzerland). The emission of the reporter gene was done in 505/535 nm. Then the normalized fluorescence showed how much of the cells have become recombinant.
  • Bioreactor cultivation- the bioreactor cultivation is important to obtain the gene ectoine gene in sufficient amounts. The predefined cultures of E. coli DH5 (Pask-ectABC) were inserted in a shakable flask with the medium containing ampicillin at 30degree Celsius and 120rpm. The bacteria in the bioreactor grows exponentially and it is always beneficial to do batch culture in Biostat Sartorius BBI System GmbH, Melsungen, Germany with the volume of 1.5 liters which will be batch culture working volume at 30degree and pH of 7-7.5 and the bioreactor cultivation is also maintained by adding 2.7 M NH4OH or 0.1 MH2SO4. The speed of the stirrer was around 1000rpm and the rate of circulation of air is 3 liter per 1minute at optimum room temperature (Marraccini et al., 1997).
  •  Analysis- With the spectrometer analysis at 700nm, dry biomass of the bacterium is checked. The concentration carbon source (which is required for the TCA cycle to run ) glucose is quantified using HPLC using the Nucleosil carbohydrate column with 0.01 N-sulfuric acid as isocratic eluent which is done at 70 degree Celsius. To measure this refractive index system was used. The concentration of ectoine is measured using isocratic gradient HPCL with the NH2 column at the same temperature with acetonitrile in the mobile phase with a flow rate of 20ml/minute and the detection of ectoine was done at 225nm by the UV detection system. Pulsed Amperometric analysis was done for all the protein molecules of ectoine as well as other solute quantification was done by HPLC. Supernatant proteins were tested with bovine serum albumin as the standard protein (Rath et al., 2009).
  • Protein sequencing and identification of Ectoine Transformants- the transforming cells extracts of S.elongatus was extracted out and put in the SDS -PAGE ( the protein was dipped in acetone). Coomassie blue stain was provided in the gel and the dried under air that is unheated. Mass spectrometry is performed for the identification of proteins in the MS-MS mode with MALDI/TRAP - XCT mass spectrometer. Act A is identified by the acylation assay by using coupled spectrometry and detection at 425nm (Shaw et al., 2013).

Conclusion on Molecular Biotechnology

This is a theoretical setup, which is designed based on experiments of different scientists all across the world. This construct might show a potential transformation in Synechococcus elongatus and in the similar way there can be chances of high infidelity in the plasmid expression vector. This experiment acknowledges all the important steps used for transforming a model bacterium.

References for Molecular Biotechnology

Cánovas, D., Vargas, C., Iglesias-Guerra, F., Csonka, L. N., Rhodes, D., Ventosa, A., & Nieto, J. J. (1997). Isolation and characterization of salt-sensitive mutants of the moderate halophile Halomonas elongata and cloning of the ectoine synthesis genes. Journal of Biological Chemistry, 272(41), 25794-25801.

Golden, S. S., & Steams, G. W. (1988). Nucleotide sequence and transcript analysis of three photosystem II genes from the cyanobacterium Synechococcus sp. PCC7942. Gene, 67(1), 85-96.

Hirokawa, Y., Kubo, T., Soma, Y., Saruta, F., & Hanai, T. (2020). Enhancement of acetyl-CoA flux for photosynthetic chemical production by pyruvate dehydrogenase complex overexpression in Synechococcus elongatus PCC 7942. Metabolic engineering, 57, 23-30.

Marraccini, P., Bulteau, S., Cassier-Chauvat, C., Mermet-Bouvier, P., & Chauvat, F. (1993). A conjugative plasmid vector for promoter analysis in several cyanobacteria of the genera Synechococcus and Synechocystis. Plant molecular biology, 23(4), 905-909.

Ono, H., Sawada, K., Khunajakr, N., Tao, T., Yamamoto, M., Hiramoto, M., ... & Murooka, Y. (1999). Characterization of biosynthetic enzymes for ectoine as a compatible solute in a moderately halophilic eubacterium, Halomonas elongata. Journal of bacteriology, 181(1), 91-99.

Rath, A., Glibowicka, M., Nadeau, V. G., Chen, G., & Deber, C. M. (2009). Detergent binding explains anomalous SDS-PAGE migration of membrane proteins. Proceedings of the National Academy of Sciences, 106(6), 1760-1765.

Rouches, M., & Lambert, G. (2020). Construction and Characterization of a Tunable Plasmid Copy Number System. Bulletin of the American Physical Society, 65.

Sato, M., Nimura-Matsune, K., Watanabe, S., Chibazakura, T., & Yoshikawa, H. (2007). Expression analysis of multiple dnaK genes in the cyanobacterium Synechococcus elongatus PCC 7942. Journal of bacteriology, 189(10), 3751-3758.

Sengupta, S., Jaiswal, D., Sengupta, A., Shah, S., Gadagkar, S., & Wangikar, P. P. (2020). Metabolic engineering of a fast-growing cyanobacterium Synechococcus elongatus PCC 11801 for photoautotrophic production of succinic acid. Biotechnology for Biofuels, 13, 1-18.

Shaw, J. B., Li, W., Holden, D. D., Zhang, Y., Griep-Raming, J., Fellers, R. T., ... & Brodbelt, J. S. (2013). Complete protein characterization using top-down mass spectrometry and ultraviolet photodissociation. Journal of the American Chemical Society, 135(34), 12646-12651.

Zhang, L., Chen, L., Diao, J., Song, X., Shi, M., & Zhang, W. (2020). Construction and analysis of an artificial consortium based on the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 to produce the platform chemical 3-hydroxypropionic acid from CO 2. Biotechnology for Biofuels, 13, 1-14.

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