LabNotebook.html
“AMAZING APRIL”
A significant month within which a series of written test and viva were conducted by various professors of our college, to select students for college’s iGEM2017 team. Finally an “AMAZING TEAM” of 13 members was formed officially named as “TEAM DEI-AGRA”!!!
“DRYLAB WORK”
The mentor along with instructors designed an entire layout and work plan for the iGEM project. Main highlights of the layout were-
1. “PROMOTER CHARACTERIZATION”- A promoter of viral origin i.e. “Pararetrovirus” and selected UTRs regions were exploited for promoter engineering to design synthetic promoters, whose cis and spacer elements were validated using online based promoter software “PLANT CARE” and “PLACE”.
2. Selection of Metal Binding Proteins: Two metal binding proteins were selected for over expression, by our designed synthetic module (promoter characterized).
1. Human Metallotheonin 3 (named as HMP3)
2. Previous iGEM Part BBa_K1478002 (named as MT4MBP)
3. Modification of iGEM Part: Previous iGEM part was modified by optimizing its codon using IDT’s Codon Bias software.
4. Primer designing: All the primers used for the cloning procedures were designed using “Primer Perfect” software.
5. bEco(Beads from E.Coli) :Conceptualized the idea of bio-bead formation [bEco] by entrapping the recombinant bacteria in sodium alginate bead. Our motivation for the concept was taken from the” “Yeast experiment for immobilization” (eng.umd.edu/~nsw/ench485/lab11.htm)
Methodical May meets juggling June
The maiden May was dedicated in learning methodologies and protocols for various lab techniques like cloning, PCR, and various lab safety procedures. Apart from this various, several unofficial teams meets were held to raise team bond and spirit.
The “methodological” May soon shifted to June, which was the month where team members were allotted different team tasks like-wiki page planning, T-Shirt designing and thus the “JUGGLING” between lab works and other project activities began!!!
“DRY LAB WORK”
The student members were engaged in-
v Taking classes by team instructors for learning biosafety rules and procedures to be followed during lab work.
v Brief introduction about project work and ideas by project mentors.
v Initiation of T-shirt designing, logo making, and wiki page.
“WETLABWORK”
The lab work was initiated with the following activities-
v Performed basic autoclaves, and observed PCR and cloning experiments that were performed by lab seniors.
v Optimized the PCR protocol for amplifying our gene of Interest from the g-block, which was delivered by IDT
v Standardized the cloning procedure for our synthetically designed promoter construct.
v The construct formations were carried out using PCR based cloning by employing the following strategy.
“Jam-packed July meets Ardent August”
These months saw the busiest schedule as team activities were in full swing from executing our outreach initiative to working on wikki page content from learning lab experiments to actually performing them. Schedule for each day was Jam-packed. The involvement in lab work increased, we developed more ardent passion for this field. During all this anxiety while awaiting lab result was at peak!!!!!!!
“WETLABWORK”
· Confirmed cloned by restriction digestion were sent for sequencing at “Department of Biochemistry” University of Delhi, South Campus, India
· After confirming the sequence, positive clones were back transformed in BL-21 strain of E.coli to obtained colonies.
· Total cellular protein was isolated from overnight grown culture of a single colony, which was subjected for quantitative and qualitative estimation.
· Bradford method was used for quantitative estimation while SDS PAGE was employed for qualitative estimation of protein samples.
Satisfactory September meets Omnipresent October
These closing months were dedicated in compilation of our lab results, which were up to our satisfaction. Then the session of being “omnipresent” started from doing final industry and farm visits, to finally gearing up for the final showdown at “Giant Jamboree” by preparing team posters, team banners, and official presentations.
“DRYLABWORK”
The final step of project; i.e. is creation of bio-beads was initiated in these months. Following activities were carried out:
1. Re-Searching publication on Bio-Beads formation.
2. Deciding the concentrations of Na-Alginate and Calcium Chloride
3. Collection of water samples from various locations having heavy metal toxicity.
4. Testing the Heavy Metal toxicity in treated as well as untreated samples by Atomic Absorption Spectrophotometry.
“WETLABWORK”
Cultures of our designed bacteria were grown overnight to obtain an optical density of “1” at 595 nm. Cells from these cultures are mixed in 1% Na-Alginate Solution. Cells were immobilized in chilled 100mM
Calcium Chloride Solution.
(Concentration of sodium alginate and calcium chloride were standardized by performing several experiments).
Efficacy Testing of Beads was performed by testing in artificially prepared toxic water having 0.5mM Concentration of CuCl2, ZnCl2, and CdCl2. For testing the efficacy 20 beads were incubated in toxic water at room temperature for 16 hours and analyzed using Atomic Absorption Spectrophotometry (AAS).
