AraC Expression effect on Pbad Promoter using TetR Promoter

Genetics

Joey Dean, Mason Clay, and Nick Hayes

Parts and Antibiotic Resistance

Pbad: BBa_K206000 pSB1C3 14A Kit Plate 3 2017

RBS-RFP: BBa_K518012 pSB1C3 18C Kit Plate 1 2017

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TetR-RBS-GFP: BBa_K624001 pSB1C3 22D Kit Plate 1 2017

AraC: BBa_K1088005 pSB1C3 15F Kit Plate 6 2017

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First Cloning

Event

Second

Cloning

Event

Transformation/DNA Isolation

We transformed all four parts into E. coli cells at one time and began working on creating our Pbad-RBS-RFP composite first

Transformations and Dna Isolations went smoothly with consistent DNA concentrations

First Cloning Event

[Pbad-RBS-RFP]

Arac (some present in cells naturally) and Arabinose form a dimer as a transcription factor that induces the promoter, Pbad.

Arabinose had to be added

Error occured plating Pbad on arabinose plates

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RBS-RFP (717bp)

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Pbad (130bp)

C-backbone [2070bp]

Second Cloning Event

[TetR-RBS-GFP-Arac-tt]

After ligation of TetR-RBS-GFP to araC-tt and the Kanamycin resistant linear backbone, the ligation was transformed and spread onto a Kanamycin plate.

An error occurred resulting in some red colonies, having no effect on the composite part colonies

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TetR-RBS-GFP

[1,571bp]

AraC

[879bp]

C-backbone [2070bp]

Analysis after Ligation

Pbad-RFP [847bp]

TetR-GFP-arac [2450bp]

Pbad-RFP digested [847bp]

Pbad-RFP undigested (Shouldn’t have used undigested Pbad-RFP)

Pbad-RFP undigested

Pbad-RFP digested

TetR-GFP-arac [2450bp]

Pbad-RFP [847bp]

K-backbone [2204bp]

TetR-GFP-arac

band is located

where it should be.

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Pbad-RFP gave skeptical results

Pbad-RFP reanalyzed with increased DNA concentrations

Pbad-RFP [847bp]

K-backbone [2204bp]

Arabinose Concentration effect on Pbad Induction

Induced Pbad-RBS-RFP was cultured in four different broths

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After 24 hours, different concentrations of arabinose were added to three broths. One broth had no arabinose as a control

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1: 0% arabinose

2: .1% arabinose

3: .2% arabinose

4. .3% arabinose

1. 2. 3. 4.

Percentages of arabinose did not change the amount of induced Pbad-rfp that was displayed

Conclusions and Lessons Learned

• Arabinose must be present in the agar to bind with Arac to induce the Pbad promoter in order for the RFP to be expressed
• Arac is present in the existing E. coli at a high enough concentration to form the dimer with Arabinose to induce some Pbad.
• Plates must be made with agar that was made with arabinose rather than pouring arabinose onto the agar plates
• When running gel electrophoresis, doubling DNA concentrations might be required to visualize DNA bands

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Future Experiment: [Pbad-RBS-RFP-tt] + [TetR Promoter- GFP-araC-tt]

We want to see whether AraC is expressed or not through the regulation of TetR promoter by tetracycline analog. We also want to look at the AraC expression levels affecting the Pbad composite. The results will be visualized by GFP and RFP

If Pbad is inhibited by the araC the RFP would not be transcribed and there would be an absence of red but if the arabinose binds to the arac, RFP will be expressed. If the TetR composite is induced GFP will be present as well.

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Thank You!

Questions?

Acknowledgements

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Dr. Nathan Reyna

Ouachita Baptist University