Promoters

E.coli strain MG1655 with the plasmid construct containing the promoter regions and regulatory proteins fused to a gfp will be used to inoculate 5 mL of M9 glucose medium containing tetracycline (20 μg/mL) to select for the plasmid. Cultures will be grown in 18 mm x 150 mm tubes. They will then be grown for 20 hours at 37℃ while being spun at 70 RPM for aeration. The cultures will be diluted 1:100 in 5 mL of pre-warmed media at the previous conditions for approximately 4 hours. Then the optical density at 600 nm (OD600) of a 500 μL sample from each culture will be determined. Using the OD600, each sample will be diluted to 0.07 OD and grown for 1 hour at the same conditions as above.

Six 200 μL samples of transformed culture, three with inducer and three without, and one 200 μL sample of untransformed cells will be pipetted into wells. There will also be one 200 μL sample of M9 medium added to the wells as a control. These wells were then placed into the SpectraMax plate reader with the settings as stated in Table 2.

Promoters and Their Inducers (Table 1)
Insert	Promoter	Regulating Molecule	Inducer
NsrR pYeaR--GFP	pYeaR	NsrR	10 mM NaNO3
rhaR rhaS pRha--GFP	pRha	RhaR	3 mM Rhamnose
pAsr--GFP	pAsr	Acid Shock RNA	Decrease pH to 5
pLacIQ -- LacI -- PL8-UV5 -- GFP	PL8-UV5	LacI	5 mM IPTG
lambda cITS lambda pR---GFP	lambda pR	lambda cITS repressor	Increase temperature above 47°C
MerR pMerT--GFP	pMerT	MerR	0.3 μM Hg(II) ion
pYJL052W--GFP	pYJL052W	Ethanol	1.5 M EtOH

SpectraMax Settings (Table 2)
Emission Spectra	525 nm
Absorption Spectra	485 nm
Time interval	0.1 sec

Kelly, Jason R., et al. "Measuring the activity of BioBrick promoters using an in vivo reference standard." Journal of biological engineering 3.1 (2009): 1-13

Toxins

E.coli strain MG1655 with the plasmid construct containing the toxin regions controlled by a lac operon will be used to inoculate 5mL of M9 medium containing kanamycin (50 μg/mL) to select for the plasmid. Cultures will be grown in 18 mm x 150 mm tubes. They will be grown for 20 hours at 37℃ while being spun at 70 RPM for aeration. The cultures would be diluted 1:100 in 5mL of pre-warmed media at the previous conditions for approximately 4 hours. Then the optical density at 600 nm (OD600) of a 500μL sample from each culture will be determined. Using the OD600, each sample will be diluted to 0.07 OD and grown for 1 hour at the same conditions as above.

Three plates containing LB agar and 200 μM IPTG will be streaked with the strain containing the plasmid construct. One plate containing LB agar and 20 mM lactose will be streaked with MG1655 without the toxin containing plasmid. Another three plates containing LB agar with no lactose will be streaked with the toxin containing strain. After 24 hours, the number of colony forming units on each plate will be counted.

NEB 2-Step PCR

Denature at 98℃ for 30 seconds.
25-35 cycles of 98℃ for 10 seconds followed 10 seconds at the annealing temperature according to the Gibson Table
Final extension at annealing temperature for 2 minutes.

Biolabs, N. (n.d.). PCR Optimization (E0555). Retrieved October 27, 2020, from https://www.neb.com/protocols/2013/01/11/pcr-optimization-e0555

Gibson Table
Data is gathered from Geneious Gibson Reports.

Promoter Constructs	Forward Primer	Tm [℃] (Forward)	Reverse Primer	Tm [℃] (Reverse)	Digestion Enzyme Used
lambda cITS lambda pR---GFP	TGCGGGCCCGGTACCAGGTTTATGGCTAGCTCAGTCCTAGG	59.1	TCTCTCTCTATCTATCTAGGGCAACCATTATCACCGCCAG	59.6	StuI
MerR pMerT--GFP	CTTGCTTCTGCTTGTTAGCCTAGATAGATAGAGAGAGAGAGAGACT	42.9	GAAAGCCTGGTCAAAGAGCCTGGTACCGGGCCCG	61.6	StuI
pAsr--GFP	CAAATTGAGGGTATGACACCTAGATAGATAGAGAGAGAGAGAGACT	59.2	GCTGAATAAATTACAGCGCCTGGTACCGGGCCCG	61.6	StuI
pLacIQ -- LacI -- PL8-UV5 -- GFP	TGCGGGCCCGGTACCAGGCGTTGACACCATCGAATGGTG	59.9	TCTCTCTCTATCTATCTAGGAATTGTTATCCGCTCACAATTTCAC	58.9	StuI
NsrR pYeaR--GFP	GGTACCAGGTTCCCATCTATAATCCTCCCTGA	57.6	CTATCTAGGCGCCTGATAATTTGCATTTT	53.2	StuI
pYJL052W--GFP	TAAATTTACACACAAAACAAACCTAGATAGATAGAGAGAGAGAGAGACT	59.2	GTCGCATATCCTATTCCCCTGGTACCGGGCCCG	61.6	StuI
rhaR rhaS pRha--GFP	GGTACCAGGCGGCGAAATAGTAATCACGAGG	59.3	CTATCTAGGTACGACCAGTCTAAAAAGCGC	57.3	StuI
Toxin Constructs
T4 Holin	TTAAAGAGGAGAAAGGTACATGGCAGCACCTAGAATATCA	56.6	CACCTTTAGACATGGTACTTATTTAGCCCTTCCTAATATTCTGGC	59.1	KpnI
hokD	TTAAAGAGGAGAAAGGTACATGAAGCAGCAAAAGGCGATG	60.1	CACCTTTAGACATGGTACTTACTCCTCAGGTTCGTAAGCTG	59.8	KpnI
parE	TTAAAGAGGAGAAAGGTACATGACGCGCAGGCTGC	60.9	CACCTTTAGACATGGTACATTACTCGAAGGTGCGGCC	60.2	KpnI
ghoT	GAAAGGTACATGGCCTTGTTCAGCAAAATCT	59.1	ACATGGTACTCAGAACAAGCTGAACAACAAGG	59.6	KpnI
Barnase	ACCTTTACAAAAATCAGATAAGTACCATGTCTAAAGGTGAAGA	55.2	GATAACCTGTGCTGCCATGTACCTTTCTCCTCTTTAATGAATTCGG	60.2	KpnI
NucD	TTAAAGAGGAGAAAGGTACATGCGACATCGCGGAGGAAA	62.6	CACCTTTAGACATGGTACTCATGAGGCAATGCCCTGC	55.0	KpnI
ccdB	TTAAAGAGGAGAAAGGTACATGCAGTTTAAGGTTTACACCT	55.9	CACCTTTAGACATGGTACTTATATTCCCCAGAACATCAGGT	56.7	KpnI
mazF	TTAAAGAGGAGAAAGGTACATGGTGAGCCGCTATGTGC	60.5	CACCTTTAGACATGGTACTTAGCCAATCAGCACGTTAA	55.4	KpnI

Gibson Procedure

Use the fragment assembly protocol from the HiFi DNA Assembly table. Each insert must be at least twice the vector. For this reaction – Vector = 0.05 pmol and each Insert = 0.1 pmol. Allowing the total reaction to be under 0.5 pmol. Reagent Volume (µl) pSB1C3 backbone: 2.35 BBa_K2797002: 5.74 Master Mix: 8.09 Add the fragments and vector to a PCR tube with the master mix and water if required. Incubate samples on a heat block 60 minutes at 50 °C.

Biolabs, N. (n.d.). NEBuilder® HiFi DNA Assembly. Retrieved October 27, 2020, from https://www.neb.com/applications/cloning-and-synthetic-biology/dna-assembly-and-cloning/nebuilder-hifi-dna-assembly

Mach1 OneShot Chemical Transformation Protocol

Pellet 1–5 ml bacterial overnight culture by centrifugation at >8000 rpm (6800 x g) for 3 min at room temperature (15–25°C). Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min. If using LyseBlue reagent, the solution will turn blue. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. If using LyseBlue reagent, the solution will turn colorless. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge. Apply 800 μl supernatant from step 5 to the QIAprep 2.0 spin column by pipetting. For centrifuge processing, follow the instructions marked with a triangle. For vacuum manifold processing, follow the instructions marked with a circle . Centrifuge for 30–60 s and discard the flow-through, or apply vacuum to the manifold to draw the solution through the QIAprep 2.0 spin column and switch off the vacuum source. Recommended: Wash the QIAprep 2.0 spin column by adding 0.5 ml Buffer PB. Centrifuge for 30–60 s and discard the flow-through, or apply vacuum to the manifold to draw the solution through the QIAprep 2.0 spin column and switch off the vacuum source. Note: This step is only required when using endA+ strains or other bacteria strains with high nuclease activity or carbohydrate content. Wash the QIAprep 2.0 spin column by adding 0.75 ml Buffer PE. Centrifuge for 30–60 s and discard the flow-through, or apply vacuum to the manifold to draw the solution through the QIAprep 2.0 spin column and switch off the vacuum source. Transfer the QIAprep 2.0 spin column to the collection tube. Centrifuge for 1 min to remove residual wash buffer. 10.Place the QIAprep 2.0 column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM TrisCl, pH 8.5) or water to the center of the QIAprep 2.0 spin column, let stand for 1 min, and centrifuge for 1 min.

Mach1 Competent Cells: Thermo Fisher Scientific - US. (n.d.). Retrieved October 27, 2020, from https://www.thermofisher.com/us/en/home/life-science/cloning/competent-cells-for-transformation/competent-cells-strains/mach1-competent-cells.html

Agarose Gel Electrophoresis

Measure 1 g of agarose. Double to obtain 2% agarose gel Add agarose to 100 mL 1x TAE buffer in a microwavable flask. TBE can substitute for TAE. Microwave 1-3 minutes until completely dissolved. Take it out and swirl every 30-45 seconds. Beware surprise boiling. Stop microwaving as soon as boiling occurs; excessive evaporation will change the agarose percentage. Don't let it boil over. Cool to about 50 °C (about when it is comfortable to keep your hand on the flask), about 5 minutes. Add ethidium bromide (EtBr) to a final concentration of xxx (usually about 10 μl of lab stock solution per 100 mL gel). EtBr binds to the DNA and allows you to visualize the DNA under ultraviolet (UV) light. Pour slowly into a gel tray with the well comb in place. Pour slowly to avoid bubbles which will disrupt the gel. Any bubbles can be pushed away from the well comb or towards the sides/edges of the gel with a pipette tip. Place newly poured gel at 4 °C for 10-15 mins OR let sit at room temperature for 20-30 mins, until it has completely solidified. Add a loading buffer to each of your DNA samples. Loading buffer serves two purposes:

one, it provides a visible dye that helps with gel loading and allows you to gauge how far the DNA has migrated, two it contains a high percentage of glycerol that increases the density of your DNA sample causing it to settle to the bottom of the gel well, instead of diffusing in the buffer. Once solidified, place the agarose gel into the gel box (electrophoresis unit). Fill the gel box with 1xTAE (or TBE) until the gel is covered. Carefully load a molecular weight ladder into the first lane of the gel. Carefully load your samples into the additional wells of the gel. Run the gel at 80-150 V until the dye line is approximately 75-80% of the way down the gel. A typical run time is about 1-1.5 hours, depending on the gel concentration and voltage. Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box. Using any device that has UV light, visualize your DNA fragments. The fragments of DNA are usually referred to as ‘bands’ due to their appearance on the gel.

Agarose Gel Electrophoresis. (n.d.). Retrieved October 27, 2020, from https://www.addgene.org/protocols/gel-electrophoresis/

Team:OhioState/Experiments