Team:IIT-Madras/Notebook
NOTEBOOK
2019
MAY
Week 3
Glycerol stock of Fusarium solani was collected from Professor Smita Srivastava’s lab.
We attempted making Potato Dextrose Agar (PDA) from scratch using potato infusion dextrose and agar. We poured slants with this media and inoculated Fusarium solani on them.
We attempted making Potato Dextrose Agar (PDA) from scratch using potato infusion dextrose and agar. We poured slants with this media and inoculated Fusarium solani on them.
MAY
Week 4
More slants were inoculated.
We sought help from Professor S.N. Gummadi and his lab members about fungal transformation.
We made glycerol stocks of the fungal spores and stored them at -80 degrees.
We sought help from Professor S.N. Gummadi and his lab members about fungal transformation.
We made glycerol stocks of the fungal spores and stored them at -80 degrees.
JUNE
Week 1
Several glycerol stocks of F. solani were made and stored.
JUNE
Week 2
We discussed the protocol for fungal transformation with Professor S.N. Gummadi and Professor Smita Srivastava and decided upon using an electroporation mediated transformation
We prepared the solutions required for transformation.
We prepared the solutions required for transformation.
JUNE
Week 3
We inoculated TOP10 containing pCAMBIA 1301 for plasmid isolation.
We discussed the usage of the enzyme beta-glucuronidase in fungal transformation and sought help with plasmid isolation from Professor Nitish R Mahapatra.
With the help of Vikas and Dhanya from Professor Nitish’s lab, we isolated pCAMBIA 1301.
We discussed the usage of the enzyme beta-glucuronidase in fungal transformation and sought help with plasmid isolation from Professor Nitish R Mahapatra.
With the help of Vikas and Dhanya from Professor Nitish’s lab, we isolated pCAMBIA 1301.
JUNE
Week 4
Khwajah from Professor Smita Srivastava’s lab suggested that we incubate slants for 7 days, collect the spores, grow them in liquid medium and then perform transformation.
We did a second round of plasmid isolation.
We poured slants, inoculated them with spores and incubated them for 7 days.
We did a second round of plasmid isolation.
We poured slants, inoculated them with spores and incubated them for 7 days.
JULY
Week 1
We discussed plasmid design with Vikas from Prof. Nitish’s lab. We inoculated the spores in liquid broth. We plated E. coli TOP10 for iGEM REC team.
Constructs of STR and GFP were designed and ordered from Twist Bioscience and IDT.
Constructs of STR and GFP were designed and ordered from Twist Bioscience and IDT.
JULY
Week 2
We performed electroporation based transformation and plated the transformants using kanamycin as the selectable marker. The control plate had no DNA.
There was growth on all the plates. However, kanamycin cannot be used a selectable marker for transformation in fungi as it’s expressed under a promoter specific to E. coli.
We corrected this by using hygromycin as a selectable marker in all further transformations.
There was growth on all the plates. However, kanamycin cannot be used a selectable marker for transformation in fungi as it’s expressed under a promoter specific to E. coli.
We corrected this by using hygromycin as a selectable marker in all further transformations.
JULY
Week 3
Another liquid culture was inoculated for transformation. Transformants were selected for on hygromycin plates. These colonies were isolated and streaked on plates for GUS staining to confirm transformation.
JULY
Week 4
We checked whether the usage of beta-glucuronidase would improve the efficiency of transformation. We sought inputs from Prof. Subramaniam on this.
The next round of transformation was conducted in two batches - with and without beta-glucuronidase. This time the transformants were directly plated for GUS staining.
The next round of transformation was conducted in two batches - with and without beta-glucuronidase. This time the transformants were directly plated for GUS staining.
AUG
Week 1
Fungal plasmid isolation was performed.
Gel electrophoresis was performed with the isolated plasmids. The gel didn’t show anything apart from the ladder.
We repeated transformation with beta-glucuronidase as the previous transformation failed. We plated on hygromycin.
We inoculated fungi for another round of transformation.
Gel electrophoresis was performed with the isolated plasmids. The gel didn’t show anything apart from the ladder.
We repeated transformation with beta-glucuronidase as the previous transformation failed. We plated on hygromycin.
We inoculated fungi for another round of transformation.
AUG
Week 2
We realised that we failed to add SDS in our previous plasmid isolation from fungi which resulted in no lysis.
Another culture inoculated for transformation.
Another culture inoculated for transformation.
AUG
Week 3
Transformation was carried out again.
We prepared reagents for another transformation.
We isolated plasmid from a transformed fungal culture using a different protocol under Khwajah's guidance.
We prepared reagents for another transformation.
We isolated plasmid from a transformed fungal culture using a different protocol under Khwajah's guidance.
AUG
Week 4
We ran a gel with our isolated plasmid samples. It was empty.
We performed another round of plasmid isolation and did not succeed.
The ordered parts arrived! Hurrah!
We performed another round of plasmid isolation and did not succeed.
The ordered parts arrived! Hurrah!
SEPT
Week 1
Our constructs were designed to compatible with Gibson assembly and restriction based cloning. There is no lab in the institute to guide us with Gibson assembly, so we decided to clone the parts into pCAMBIA 1301 using restriction enzymes.
Prof. Nitish's lab guided us with cloning. Abrar, Anand, Dhanya, and Vikas guided us.
Prof. Nitish's lab guided us with cloning. Abrar, Anand, Dhanya, and Vikas guided us.
SEPT
Week 2
We isolated plasmids for digestion using a mini-prep.
We set up restriction digestion for GFP (using PstI and AflII), STR (using EcoRI and PstI) and pCAMBIA 1301 (using AflII and EcoRI).
The GFP and STR fragments were ligated using a T4 ligase. The fragment was then ligated with the cut plasmid.
The ligation mixture was transformed into competent TOP10 cells.
We got one colony. One colony!
We ordered primers for GFP and STR to amplify the parts.
We set up restriction digestion for GFP (using PstI and AflII), STR (using EcoRI and PstI) and pCAMBIA 1301 (using AflII and EcoRI).
The GFP and STR fragments were ligated using a T4 ligase. The fragment was then ligated with the cut plasmid.
The ligation mixture was transformed into competent TOP10 cells.
We got one colony. One colony!
We ordered primers for GFP and STR to amplify the parts.
SEPT
Week 3
The colony was inoculated in 5ml of LB and plasmid isolation was performed from 2ml of this culture. The isolates were run on a gel but sadly, no bands were observed.
Cloning had failed and we had to rethink our strategy.
Cloning had failed and we had to rethink our strategy.
SEPT
Week 4
We redid plasmid isolation from E.coli transformed with our part from the remaining liquid culture. A smear was observed on the gel.
Our anxiety peaked while waiting for primers to arrive.
Our anxiety peaked while waiting for primers to arrive.
OCT
Week 1
Primers arrived!
We reconstituted primers and set up a gradient PCR for GFP and STR to find the annealing temperatures.
PCRs for GFP and STR were set up using the annealing temperatures obtained. These were purified using a PCR cleanup.
We used the same cloning strategy and repeated restriction digestion, ligation and transformation.
This failed.
We had a fruitful discussion with Prof. Subramaniam and Prof. Nitish who helped us redesign our cloning strategy.
We reconstituted primers and set up a gradient PCR for GFP and STR to find the annealing temperatures.
PCRs for GFP and STR were set up using the annealing temperatures obtained. These were purified using a PCR cleanup.
We used the same cloning strategy and repeated restriction digestion, ligation and transformation.
This failed.
We had a fruitful discussion with Prof. Subramaniam and Prof. Nitish who helped us redesign our cloning strategy.
OCT
Week 2
We started all the procedures for cloning from the start. Fun times.
Our plasmid quality was low, so we isolated plasmid using a midi-prep. GFP was amplified and gel purified.
We digested GFP and pCAMBIA 1301 followed by ligation and transformation into ultra-competent DH5α cells.
Our plasmid quality was low, so we isolated plasmid using a midi-prep. GFP was amplified and gel purified.
We digested GFP and pCAMBIA 1301 followed by ligation and transformation into ultra-competent DH5α cells.
OCT
Week 2
Success!
We had over 25 colonies. These were inoculated in LB and plasmids were isolated. This was confirmed by running a gel.
These plasmids were subjected to restriction digestion to observe an insert release.
This part of GFP in pCAMBIA 1301 under the control of a CaMV35s promoter was validated by fluorometry studies.
We had over 25 colonies. These were inoculated in LB and plasmids were isolated. This was confirmed by running a gel.
These plasmids were subjected to restriction digestion to observe an insert release.
This part of GFP in pCAMBIA 1301 under the control of a CaMV35s promoter was validated by fluorometry studies.
OCT
Week 3
We characterised a CaMV35s promoter in E. coli using a hph gene attached to the promoter. The MIC of E. coli TOP10 was found to be 50µg/ml. TOP10 transformed with the above part was resistant to up to 200µg/ml of Hygromycin.
Contact Us
Department of Biotechnology, IIT Madras.
Chennai 36, India.
Sponsors
