Difference between revisions of "Part:BBa J13002:Experience"

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Part J13002: Characterization by UMN

In 2009, the University of Minnesota's iGEM team chose to characterize this part. We were working on engineering a logical AND gate for the iGEM competition and were interested in existing parts that had the same regulators. We used MIT 2004's exemplar characterization of Part F2620 as a template for our characterization and characterized this part in terms of Transfer Function and Stability.

Device Design

Since this part already contained an RBS, we attached GFP, included in the 2009 iGEM Parts Kit, to the promoter and RBS and examined the fluorescence, which is directly proportional to [http://parts2.mit.edu/wiki/index.php/Abstraction_hierarchy_and_PoPS Polymerase Per Second (PoPS)]. We inserted these two parts into the plasmid backbone pSB3k3 and induced the resulting device at increasing concentrations of aTc: 0, 1, 10, 50, 100 and 200 ng/ml.

Results

Transfer Function

As you can see on the graphs above, there was a general upward trend of GFP production with the concentration of aTc. The error bars in the graphs represent the standard error. Interestingly, we observed a spike in GFP production at 50 ng/ml of aTc. During the sampling, the culture growing in 50 ng/ml aTc inducer media did not grow as fast as the others, with the OD595 as low as 0.08 rather than the preferred 0.2. We continued sampling at each hour point for this concentration bu did not see obvious pellets during the resuspension in PFA or PBS. Since the inducer is an antibiotic, we hypothesized that it could be having a toxic effect on the cell and subsequent GFP production. We did perform this experiment again but did not see any induction.

Stability

We also examined the stability of the device, which is how the production of GFP changes for each inducer concentration over multiple rounds of cell division. As you can see from the graphs above, the device is clearly inducible with a general downward trend in GFP production over time. The graph of aTc concentration of 50 ng/ml again demonstrates the spike in GFP production that we observed at this concentration. This is the optimal inducer concentration for the device.

Protocol

1. Resuspend DNA for GFP and the promoter + RBS from the 2009 iGEM Parts Kit in 15 ul water
2. Transform DNA into Top10 Chemically Competent Cells
3. Pick colonies from transformation and grow in 2 ml cultures with appropriate antibiotic
4. Prep the plasmids
5. Determine DNA purity
6. For a further check, may perform PCR and run the products out on an agarose gel to see if amplification was successful. Use forward and reverse primers for BioBricks.
7. Sequence plasmids
8. https://static.igem.org/mediawiki/2009/6/68/Restriction_Digest.pdf Digest] the promoter, GFP and plasmid backbone with the appropriate restriction enzymes for later ligation
9. [https://static.igem.org/mediawiki/2009/a/a8/DNA_Purification.pdf Gel purify digest products
10. Ligate the promoter + RBS and GFP after restriction enzyme digest in the plasmid backbone pSB3K3 overnight
12. Sequence ligation products
14. Transform the ligation products into DH5alphaPro cells, which express TetR
15. Pick colonies and grow in 2ml liquid cultures plus kanamycin
16. Make up 5ml stock of inducer media at 0, 1, 10, 50, 100, 200 ng/ml aTc and one batch of 0.5 ml inducer media with twice the aTc concentration for the first induction
17. Spec cells at time 0
18. Inoculate 2x [aTc] media with 0.5 ml cells
19. Every hour, spec the DNA and, based on the OD595, which should be 0.2 dilute the cultures with inducer media and take 200 ul for sampling
20. Spin down 200 uL cells at 5K rpm for 5 minutes so as not to squash them
21. Remove supernatant and resuspend in 0.5 ml 4% sterile filter PFA
22. After 30 minutes, the cells have been fixed and can be spun down at 5K rpm for 5 minutes
23. Resuspend cells in 0.5 ml PBS and store in fridge until ready for flow cytometry
24. Sample for 9 hours
25. Run through flow cytometer and analyze using FlowJo software and Excel spreadsheet




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