Team:CSU Fort Collins/Results

COLORADO STATE
UNIVERSITY iGEM

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Project Results

Contents

1 Key Results
2 Breakdown
3 Trans-zeatin Production
4 Kill Switch
5 References

Key Results

Key Achievements

• Construction and submission of multiple team parts
• Proof of the composite lac promoter:fadD:fadL part function
• Characterization of KillerRed in E. coli for use as an induced lethality switch

Key Challenges

• Reworking breakdown construct ideas after fadD experiment
• Inconclusive results about trans-zeatin production
• Proper handling and experimental design for testing KillerRed

Breakdown Results

Trans-zeatin Production Results

Our team was able to successfully create two constructs, a basic part and a composite part which included an inducible promoter, which encode for the trans-zeatin biosynthesis pathway. We ran multiple growth experiments to assess how much metabolic stress, if any, our construct introduced into our strain. We tested our strain, with the tzS:LOG operon behind a lac promoter, against a plasmid just containing the lac promoter. We compared their growth over 24 hours and 72 hours in shake flasks, as well as in bioreactors over 72 hours. From the results, we can conclude that the presence of the genes does not increase metabolic stress on the cell.


Figure 1: Growth comparison between a control strain and our trans-zeatin strain in shake flasks over 24 hours.


Figure 2: Growth comparison between a control strain and our trans-zeatin strain in shake flasks over 72 hours.


Figure 3: Growth comparison between a control strain and our trans-zeatin strain in bioreactors over 72 hours.

Throughout these experiments, we also collected suspension samples which we purified and ran on a reverse-phase HPLC column. Initially, we extracted using 80% methanol and syringe filtration. The HPLC results had too much background to be conclusive, so we developed a new method, based on extraction of zeatin from coconut water^[1]. The procedure required single-phase extraction (SPE) using C-18 HyperSep columns. Again, the results showed a lot of background, but no conclusive evidence of trans-zeatin production. The HPLC comparisons show no noticeable peak differences at the time of the retention standards.


Figure 4: HPLC results. A) Bioreactor lac promoter control after 72 hours B) Bioreactor trans-zeatin strain after 72 hours C) Trans-zeatin time retention standard D) Shake flask lac promoter control after 72 hours E) Shake flask trans-zeatin strain after 72 hours F) Trans-zeatin time retention standard. There is no noticeable difference in peaks between the controls and the test strains, and there are no noticeable peaks present at the time of the retention standard.

There are two possible reasons for these results. It is possible that trans-zeatin is being created, but is below our limit of detection using HPLC-UV. To resolve this, we will use a more sensitive process, liquid chromatography with dual mass spectrophotometry, which can detect trans-zeatin presence in the pico- to nanogram per mL range. Alternatively, it is possible that the E. coli is not producing trans-zeatin at all. To determine if this is the issue, we will run qPCR to detect mRNA expression.

Kill Switch Results

References

1. Ma, Zhen et al. "Simultaneous analysis of different classes of phytohormones in coconut (Cocos nucifera L.) water using high-performance liquid chromatography and liquid chromatography–tandem mass spectrometry after solid-phase extraction." Analytica Chimica Acta 610:274-281. January 2008.

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