UNL 2018 Improving Early Detection of The Emerald Ash Borer

Notebook

Detailed protocols can be found on our experiments page.

Complete notebook can be found on this page: NOTEBOOK

Week 1 – June 4th-8th:

New team members went through a two-week training program. The team learned how to make LB Broth and agar plates. Training was done on how to prepare chemically competent cells and the respective transformation. PCR for mCherry gene was performed and then analyzed using gel electrophoresis.

Week 2 – June 11th-15th

Training was continued till the end of the week. DNA recovery was performed on the PCR products. Training was done on molecular cloning techniques SLIC and Subcloing.

Week 3 – June 18th-22nd

The third week consisted mainly of InterLab calibrations. We received and streaked DH5-α E. coli hosting plasmids 100167, 100168, and 100169 separately. These plasmids were purchased from Addgene and are the basis of our experiments.

Week 4 – June 25th-29th

The bulk of the InterLab study was conducted by Rachel and Drew. Initial minipreps and transformations into GeneHog E. coli were performed by Gabe.

Week 5 – July 2nd-6th

Two fragment-Sequence and Ligation Independent Cloning (SLIC) was led by Gabe and advised by Jarek. The first attempt was unsuccessful. Second attempt was initiated. Glycerol stocks of the original order were made by Rachel.

Week 6 – July 9th-13th

The team prepares protocol for sesquiterpene assay(s) and starts preliminary in vivo experiment on the originally ordered plasmids, 100167 and 100169. Second SLIC attempt failed due to biological contamination. (Link to worst gels). Third SLIC attempt completed and sent for sequencing.

Week 7 – July 16th-20th

Initial sequencing looks good. More primers are ordered to sequence middle of inserted gene. With the help of DJ, Nic and the team prepared GC-MS samples from last week’s experiment. The goal was to detect amorphadiene, but it did not show on the resulting chromatograms. Restriction digest and PCR was used to help verify a successful cloning. Glycerol stock was made of the double plasmid transformation (100167 and 100169-TPS4).

Week 8 – July 23rd-27th

The sequencing of the cloned plasmid was confirmed. A new assay was carried out, led by Rachel. Three cultures were used: 2 plasmid system with pADS with IPTG induction, 2 plasmid system with pTPS4 with IPTG induction, and a GeneHog control. Nic performed ligation cloning for the codon optimized variant of TPS4. Transformation looks successful.

Week 9 – July 30th-August 3rd

PCR was used on the most recent clone. The PCR product was sent in for sequencing. Rachel prepared GC-MS samples and analyzed the results. The samples from the ADS cultures showed presence of amorphadiene. The samples from the pTPS4 cultures were inconclusive. A glycerol stock of a pTPS4-CO and 100167 double transformation was made by Rachel. The sequencing for the pTPS4-CO plasmid was later confirmed.

Week 10 – August 6th-10th

Drew made first batch of Terrific Broth. Terrific Broth will be used for the rest of the assays. Our third assay (reported in results as assay 1 since it was the first assay with pTPS4-CO) was carried out with three different cultures: 2 plasmid system with pADS, 2 plasmid system with pTPS4, and 2 plasmid system with pTPS4-CO. Cell pellets and the remaining octane layer were saved.

Week 11 – August 13th-17th

GC-MS samples were prepared and run by Rachel and Howard. This week Drew attempted SDS-PAGE. All attempts failed. Future work will look into protein expression.

Week 12 – August 20th-24th

Our fourth assay (reported in results as assay 2) was ran this week led by Rachel and Nic. Cell pellets and the remaining octane layer was saved. Classes begin at UNL.

Week 13 – August 27th-31st

Another assay, the fifth (known as assay 3), was ran this week. Caryophyllene started to be used as an internal standard in GC-MS analysis to quantify sesquiterpene production.

Week 14 – September 3rd-7th

Re-analysis of previous assay samples using the internal standard failed. Made and tested a different internal standard concentration. Re-analyzed two sample sets with new internal standard.

Week 15 – September 10th-14th

This week and every week after, Rachel has worked on GC-MS sample prep and analysis.

Week 16 – September 17th-21st

Nic begins work to clone the TPS4-CO coding sequence into the iGEM submission vector, pSB1C3. Initial ligation for cloning failed.

Week 17 – September 24th-28th

Sixth and final assay (reported as assay 4 in results) was performed. Last attempted SDS-PAGE by Drew, resulting in another failure. The ligation reaction was corrected; transformation was a success. Restriction digest was positive.

Week 18 – October 1st-5th

Initial sequencing was promising, but didn’t run the full length of the gene. Follow-up sequencing was performed and confirmed a positive cloning.

Week 19 – October 8th-12th

Nic ran miniprep for the new pSB1C3_TPS4-CO. Plenty of DNA was pipetted into the 96 well submission plate and dried overnight. Our part was submitted by Nic on the 9th and the package was successfully delivered to iGEM HQ on the 15th.

Week 20 – October 15th-19th

All wet lab work is completed by this point. The team is frantically trying to finish the wiki.

Team:UNebraska-Lincoln/Notebook