Team:Cadets2Vets/Notebook September

Briefly centrifuge tubes to pellet bacteria and decant media so that only ~100 µL is left in the tube. Resuspend the cells in the remaining media, and pipet onto a 25µg/mL chloramphenicol-selection agar plate.

Using a sterile spreader, spread culture evenly across the agar plate. Incubate the agar plates upside down (agar should be on top) in a 37C incubator overnight. Decontaminate the spreader by soaking it in a 10% bleach solution before discarding.

Save 500 µL of the overnight culture as a glycerol stock by adding 500 µL glycerol to it. Store in -20C temporarily and move it to -80C later.

Prepare plasmid DNA from the bacteria cultures using the Promega miniprep kit, following the manual instructions except the elution volume. Elute in 50 µL instead of 100 µL.

The Nanodrop readings for the plasmids are:

Plasmid Name	Concentration (ng/µL)	A260	A260/A280
pSB1C3/PcArsR-1	202	4.042	1.89
pSB1C3/PcArsR-2	162	3.249	1.9
pSB1C3/PArsRGFP-1	185	3.711	1.88
pSB1C3/PArsRGFP-2	214	4.289	1.85

Plasmid concentrations and purity look good, so we will proceed with checking the cloning by using restriction digest. Because we did not perform any PCR, we are not worried about DNA mutations.

The next restriction digest will have 2 functions: 1) to check whether we inserted the correct insert into the vector and 2) to create new inserts that we will ligate together to make the complete Ars2.0 circuit. We will use Genome Compiler to perform an in silico digest and determine the fragment sizes we can expect.

Plasmid

Restriction Enzymes

Expected Fragment Size

pSB1C3/PcArsR

EcoRI-HF and SalI

Vector: 2070bp

Insert: 550bp

pSB1C3/PArsRGFP

SalI and PstI

Vector: 2070bp

Insert: 950bp

Restriction digest mix: Restriction digest mix:

Reagent	Vol for 1 rxn (µL)	Vol for 2.4 rxns (µL)	Reagent	Vol for 1 rxn (µL)	Vol for 2.4 rxns (µL)
10X Buffer 2.1	2.5	6	10X Buffer 2.1	2.5	6
EcoRI-HF	0.05	0.12	SalI	0.05	0.12
SalI	0.05	0.12	PstI	0.05	0.12
water	16.2	38.9	water	17	38.9
PcArsR DNA	1 µg	--	PArsRGFP	1 µg	--
	To 25 µL rxn			To 25 µL rxn

Digest four tubes at 37C for 1.5hrs.

Add Gel Loading Dye Purple to digestions. This dye will have a pink dye migrate at 400bp and a blue dye migrate at 4000bp. Electrophorese DNA out on a 1% TAE agarose gel containing SybrSafe DNA Gel Stain. Load 0.5ug of Thermo GeneRuler 1Kb Plus DNA ladder to the first well, then load the DNA into subsequent wells. Electrophorese gel at 120V for ~35min.

DNA fragments look exactly as predicted. Success! Now we will ligate these inserts together to make Ars2.0. To extract the DNA fragments we want out of the gel, we inserted filter paper backed by a dialysis membrane below each desired fragment. The DNA will migrate into the filter paper, and be stopped by the dialysis membrane which won’t allow molecules bigger than 8kDa past it. We checked the migration of DNA using the UV imager (gel on right; visible bands are the vector backbone).

Then we removed the filter paper from the gel and placed each one in a 0.5mL microcentrifuge tube that had a hole pierced in the bottom. We nested these tubes inside 1.5mL microcentrifuge tubes and centrifuged them briefly to force the buffer containing DNA out of the filter paper. We now have the desired fragments cut with complementary enzymes that will let us join these two Parts together with the previously digested pSB1C3.

First, we ligated the two Parts together. Because PArsRGFP is twice as big as PcArsR, we used twice as much volume to approximate an equal molar ratio. Incubate Ligation Step 1 at 16C for 1hr. Then we will prepare a ligation to add the backbone vector. For Ligation Step 2, we will make a negative control of adding water instead of insert.

Ligation Step 1 mix: lllllllllllllllllllllllllllllllllllll Ligation Step 2 mix:

Reagent	Vol for 1 rxn (µL)	Reagent	Vol for 1 rxn (µL)	Vol for 2.2 rxns (µL)
10X T4 Ligase Buffer	1	10X T4 Ligase Buffer	1.5	3.3
T4 Ligase	0.5	T4 Ligase	0.5	1.1
water	1	water	4.5	9.9
Insert PcArsR	2.5	insert or water	7.5	16.5
Insert PArsRGFP	5
	To 10uL		To 15 µL

For Ligation Step 2, make the entire MM to ligate with water. Leave 15 µL for the negative control, and then take 5 µL of MM and add to the Ligation Step 1. Ligate at 16C overnight.

Nanodrop readings of the plasmids:

Plasmid Name	Concentration (ng/µL)	A260	A260/A280
pSB1C3/Ars2.0-1	129	2.584	1.88
pSB1C3/Ars2.0-2	131	2.626	1.88

Pour 1% TAE gel. Electrophorese 120V for 15min.

Band is faint but PcArsR fragment is visible at 500bp for both clones. Interestingly, the vector backbones are different sizes. The expected size was 3000bp but Clone 2 is only 2000bp. This indicates that Clone 1 contains pSB1C3+PArsRGFP while Clone 2 only contains pSB1C3.

Clone 1 has the correct fragment pattern and therefore contains both inserts in the correct orientation. pSB1C3/Ars2.0 cloning is complete.

Team:Cadets2Vets/Notebook September

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