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iGEM 2021 Contributions

BBa_I716155

hemD

During heme biosynthesis, this enzyme catalyses the asymmetric cyclization of linear tetrapyrrole to form a uroporphyrinogen III isomer. At the catalytic site, hydroxymethylbilane use as a substrate gives uroporphyrinogen III.1 Guocheng Du et al. group identified that along with hemA and hemL, upregulation of hemD and hemF increases the 5-Aminolevulinic acid accumulation which is a promising source for cancer treatment.2

References

  1. Schubert, H. L., Phillips, J. D., Heroux, A., & Hill, C. P. (2008). Structure and Mechanistic Implications of a Uroporphyrinogen III Synthase−Product Complex. Biochemistry, 47(33), 8648–8655. https://doi.org/10.1021/bi800635y
  2. Zhang, J., Kang, Z., Chen, J., & Du, G. (2015). Optimization of the heme biosynthesis pathway for the production of 5-aminolevulinic acid in Escherichia coli. Scientific Reports, 5(1). https://doi.org/10.1038/srep08584

BBa_I742107

COMT coding sequence

It has been found that strong downregulation of COMT in transgenic alfalfa plants decrease the lignin amount and a reduction in the number of units of guaiacyl (G) lignin with a total loss of syringyl (S) units in monomeric and dimeric lignin degradation products.[1]

References

  1. Guo, D., Chen, F., Inoue, K., Blount, J. W., & Dixon, R. A. (2001). Downregulation of Caffeic Acid 3-O-Methyltransferase and Caffeoyl CoA 3-O-Methyltransferase in Transgenic Alfalfa: Impacts on Lignin Structure and Implications for the Biosynthesis of G and S Lignin. The Plant Cell, 13(1), 73.

BBa_K208001

Phasin(PhaP)

Recent reports have shown that heterologous expression of phasin PhaP in E. coli increase tolerance to several biotechnologically relevant chemicals, and it enhances bacterial fitness in the presence of biofuels, such as ethanol and butanol. Also, Phasin with chaperone-like properties can increase bacterial tolerance to several biochemicals.1 Secondary-structure prediction and circular dichroism (CD) spectroscopy of PhaP revealed that the protein consists of two α-helices, both of them associating with PHB granules.2

References

  1. Schubert, H. L., Phillips, J. D., Heroux, A., & Hill, C. P. (2008). Structure and Mechanistic Implications of a Uroporphyrinogen III Synthase−Product Complex. Biochemistry, 47(33), 8648–8655. https://doi.org/10.1021/bi800635y
  2. Hauf, W., Watzer, B., Roos, N., Klotz, A., & Forchhammer, K. (2015). Photoautotrophic Polyhydroxybutyrate Granule Formation Is Regulated by Cyanobacterial Phasin PhaP in Synechocystis sp. Strain PCC 6803. Applied and Environmental Microbiology, 81(13), 4411–4422. https://doi.org/10.1128/aem.00604-15

BBa_K808027

EstA

A cold-adapted esterase was extracted from psychrotrophic bacterium Pseudoalteromonas sp. strain 643A, which is present in the alimentary tract of Antarctic krill Euphasia superba Dana. This protein consists of 206 amino acids residue with a molecular mass of 23,036 Da and belongs to the member of the GDSL-lipolytic enzymes family.1

References

  1. Cieśliński, H., Białkowska, A. M., Długołęcka, A., Daroch, M., Tkaczuk, K. L., Kalinowska, H., Kur, J., & Turkiewicz, M. (2007). A cold-adapted esterase from psychrotrophic Pseudoalteromas sp. strain 643A. Archives of Microbiology, 188(1), 27–36. https://doi.org/10.1007/s00203-007-0220-2

BBa_K777117

motB

It is an amphipathic protein with 308 long amino acid residues, having 22 residues long hydrophobic region near N-terminus. Reports have shown that membranes have limited capacity to hold this protein, and insertion of MotB in membrane requires other components, including MotA protein.

References

  1. Stader, J., Matsumura, P., Vacante, D., Dean, G. and Macnab, R., 1986. Nucleotide sequence of the Escherichia coli motB gene and site-limited incorporation of its product into the cytoplasmic membrane. Journal of Bacteriology, 166(1), pp.244-252.

BBa_K777117

LuxEG

It is an amphipathic protein with 308 long amino acid residues, having 22 residues long hydrophobic region near N-terminus. Reports have shown that membranes have limited capacity to hold this protein, and insertion of MotB in membrane requires other components, including MotA protein.

References

  1. Meighen, E. A. (1994). Genetics of Bacterial Bioluminescence. Annual Review of Genetics, 28(1), 117–139. https://doi.org/10.1146/annurev.ge.28.120194.001001
  2. Swartzman, A., Kapoor, S., Graham, A. F., & Meighen, E. A. (1990). A new Vibrio fischeri lux gene precedes a bidirectional termination site for the lux operon. Journal of Bacteriology, 172(12), 6797–6802. https://doi.org/10.1128/jb.172.12.6797-6802.1990

BBa_J63008

SV40 nuclear localization sequence from SV40; yeast codon-optimized

The nuclear import of plasmid DNA mediated by NLS from SV40 T antigen is a two-step process. The first step involves targeting of import substance to the cytoplasmic side of the nuclear membrane. The process is energy independent and can occur at 0o C. The second step is translocation which is energy and temperature-dependent. It has been found that chelating cytosolic Ca2+ with BAPTA inhibit the DNA-NLS import, and DNA-NLS import is competitively inhibited by BSA-NLS conjugates, reinforcing the specificity of NLS in directing the nuclear import of DNA-NLS complexes.1

References

  1. Collas, P., & Aleström, P. (1996). Nuclear localization signal of SV40 T antigen directs import of plasmid DNA into sea urchin male pronuclei in vitro. Molecular Reproduction and Development, 45(4), 431–438

IISER Bhopal Parts characterization (ECFP Coding Device) in partnership with IISER Mohali

Part: BBa_J04421

Step 1:- Extracting DNA out from the kit plate (15P).

Step 2:- Extracted DNA was transformed into DH5 Alpha cells.

Result:-


Step 3:- 10 ml of primary culture was grown using 1 isolated colony, and it was grown at 37 degree C for 12 hours.

Step 4:- Primary culture is divided into two parts, one is used for plasmid isolation and other is used for secondary culture.

Step 5:- 1 percent of primary culture was used to set the secondary culture in 3 (15 ml) falcons in 5 ml media and after approximately 2.5 hours when OD reached around 0.6, IPTG was added at different concentration, 0mM, 2mM, and 5mM. After that cultures were induced at 37 degree Celsius for 12 hours.

Step 6:- 2mM Culture was pelleted, supernatant was discarded. 20 uL 8M urea was added to the pellet, pellet was pipetted until it was dissolved. 5 uL protein loading dye (containing Beta mercap) was added to it. Sample was heated for 10 minutes at 80 degrees. Samples were run on SDS gel.

Results:

The following image was obtained in the gel doc. A dark prominent band was obtained around 25 KdA. The size of ECFP was 25KdA (literature survey).


Step 7:- Fluorescence was taken for three samples (LB media, 2mM, and 5mM).

Result: - No conclusive result, control is showing higher fluorescence (maybe auto fluorescence).


Step 8 :- Protein isolation from cultures:

Materials: - Phosphate Buffer (20mM Sodium Phosphate, 50Mm Nacl), 8M Urea

Method: - Cultures were pelleted, supernatant was discarded. 200uL 8M urea was added to the pellet, pellet was pipetted until it was dissolved. 2 mL Phosphate buffer was added to it (make up the volume to level up for the fluorescence cuvette). Next step was to centrifuge it at 6500 rpm for 5 min. Now carefully pipette out the supernatant into a fresh MCT without disturbing the pellet

Step 9:- Fluorescence (0mM (ctrl), 2mM, and 5mM).

Result: - No conclusive result, control (0mM uninduced) shows similar fluorescence as induced, and 5mM IPTG shows lower fluorescence than 2mM.



Step 10: -Since DH5 alpha cells are used as cloning strain we thought to repeat all steps in Expression strain BL21 (DE3) pLysS. It also contains a plasmid, pLysS, which carries the gene encoding T7 lysozyme. T7 lysozyme lowers the background expression level of target genes under the control of the T7 promoter but does not interfere with the level of expression achieved following induction by IPTG.

Step11:- 1 ul Plasmid isolated in Step4 were transformed into strain BL21 (DE3) pLysS.

Result:-

Step 12:- Primary culture of 5 ml using 1 isolated colonies, and let it grow at 37 degree C for 12 hours.

Step 13:- 1 percent of primary culture is used for secondary culture in 4 (15 ml falcons) in 5 ml media and after approximately 2.5 hours when OD reached around 0.6, IPTG was added at different concentrations, 0mM, 3mM, 5mM. After that cultures were induced at 37 degree Celsius for 12 hours.

Step 14:- Protein was isolated using Step 8

Step 15:- Fluorescence was measured (0mM (ctrl), 3mM, and 5mM).

Result: - Correct pattern observed, control (0mM uninduced) shows lower fluorescence in comparison to the induced culture, and 5mM IPTG shows highest fluorescence. But the difference was not much.


Step 16:- Currently we are working on Expression strain BL21 (DE3) standard and BL21 Lemo21 (DE3) for clear results.

Step17:- 1 ul Plasmid isolated in Step4 were transformed in both strains.



Step 18:- Primary culture of 5 ml using 1 isolated colonies, and let it grow at 37 degree C for 12 hours.

Step 19:- 1 percent of primary culture is used for secondary culture in 3 (15 ml falcons) in 5 ml media and after approximately 2.5 hours when OD reacheD around 0.6, IPTG was added at different concentrations, 0mM, 2mM, 5mM. After that cultures were induced at 37 degree Celsius for 12 hours.

Step 20:- Protein was isolated using Step 8

Step 21:- Fluorescence was measured (0mM (ctrl), 2mM, and 5mM).

Results: Step 18:- Primary culture of 5 ml using 1 isolated colonies, and let it grow at 37 degree C for 12 hours.

Step 19:- 1 percent of primary culture is used for secondary culture in 3 (15 ml falcons) in 5 ml media and after approximately 2.5 hours when OD reached around 0.6, IPTG was added at different concentration, 0mM, 2mM, 5mM. After that, cultures were induced at 37 degree Celsius for 12 hours.

Step 20:- Protein was isolated using Step 8

Step 21:- Fluorescence was measured (0mM (ctrl), 2mM, and 5mM).



(Note:- For all the work (in plates, primary and secondary culture) antibiotic used is chloramphenicol.)