6/3/2019 - 6/7/2019

Monday

Lab Pats began by listening to safety training and engaging in a walk-through of lab facilities, including fire extinguishers, equipment, and safety locations.Wright Scholars in-processedLab Pats created and autoclaved LB Agar and engaged new members in micro-pipette training. New members also learned to use gel electrophoresis equipment. Poured plates - chloramphenicol. UES Members in-processed.

Located RhlR plasmid (glycerol stock #7 in Amy's box). Streaked LB-Chl plates and placed in incubator at 37°C overnight Began working with benchling.com program.

Thursday

Colonies successfully grew on plates. We picked colonies and created overnights with LB Broth. 2 overnights were created for each colony. One overnight would include 3 μL of the quorum sensing molecule N-butanoyl-L homoserine lactone (C4-HSL) to induce the production of the GFPa1 and the other overnight would not include C4-HSL, but DMSO, to ensure that C4-HSL was the factor responsible for the production of GFPa1.

Friday

The entire team and mentors met today at UES to discuss the project in detail, the meetup we plan on hosting June 22, and medal criteria. Afterwards, the Lab Pats headed back to lab. In lab, the team looked at the tubes from the overnights under the blue light. We had successful results. The blanks were not contaminated, the C4-HSL tubes glowed, and the DMSO tubes did not glow. We also made glycerol stocks out of plate #2 tube #2 of the DMSO (Amy's RhlR stock #7)

6/10/2019 - 6/14/19

Monday

LabPats composed an LB Broth and Chloramphenicol solution (3mL broth and 3μL antibiotic). 3 tubes were then made with 3μL C4-HSL and DMSO in respective tubes. The glycerol stock we made on 6/7/19 was put into DMSO and C4-HSL only, and the tubes were put into overnights at 37o C

Tuesday

The LabPats miniprepped the RhlR from the overnights. We followed the miniprep procedure with the goal of extracting the plasmid. The tubes were labeled RhlR 1 and RhlR 2. LabPats made overnights of both the DMSO and C4-HSL.Nanodrop Results of C4-HSL Induced RhlR Plasmid

	A	B	C
1	Tube Number	260/280 ratio	DNA concentration (ng / μL)
2	1	1.88	196.5
3	2	1.88	80.3
4	EB	0.42	0.1

Wednesday

In lab, we made more overnights that included 3 tubes of 3OC12 with sfGFP (2017 part), 3 tubes of DMSO with sfGFP, 3 tubes of C4-HSL with RhlR plasmid, 3 tubes of DMSO with RhlR plasmid, and a blank. The 3OC12 with sfGFP is the LabPats part from 2017 that we are quantifying this year. For the 3OC12 tubes, the concentration was 3μL of 10μM into 3mL of LB. The C4-HSL concentration was 3μL of 100μM into 3mL of LB. These 13 tubes were put into the incubator at 37o C overnight.The team also met up with 3 members of the USAFA iGEM team for lunch at City Barbecue.

Thursday

Lab Pats made standard curves and took measurements of the fluorescence and absorbance of the overnights from Wednesday (6/12/19). We used both the plate reader and flow cytometer to measure overall fluorescence and absorbance in the wells and individual cell absorption/fluorescence respectively. We also did a 1/2 dilution in order to fit the measurements in our standard curves.

Plate Reader
Settings for absorbance: 600 OD, top read, purple adapter for bottom, 10 seconds shaken, clear wells

Settings for fluorescence: 480-510λ, bottom read, 5 seconds shaken, black well w/ clear bottom

Green to white (A1-D12) serial dilutions for standard curve
Dark red (E1-E3) RhlR w/C4HSL

Light red (E4-E6) diluted RhlR w/C4HSL
Dark Orange (F1-F3) RhlR w/DMSO

Light orange (F4-F6) diluted RhlR w/DMSO

Dark blue (G1-G3) pLas w/ 3OC12

Light blue (G4-G6) diluted pLas w/3OC12

Dark purple (H1-H3) pLas w/DMSO

Light purple (H7-H9)
Black (H4-H6 and (H9-H12)

Flow Cytometer
LabPats used spherotech cat. no. URCP 38-2k log no. Ak02
300 μL of culture were in the tubes and then 1 mL of 1X PBS buffer was added to the tubes
There were 13 tubes total with 100 μL per sample. 1-3 was C4HSL in RhlR plasmid. 4-6 was DMSO in RhlR. 7-9 was 3OC12 in pLas. 10-12 was DMSO in pLas. 13 was the beads provided in the kit (1-2 drops)We also made a 150mL overnight from glycerol #7 of the RhlR plasmid in preparation for the maxiprep on 6/14.

Friday

The LabPats took RhlR overnights from 6/13 out of the incubator and did a MaxiPrep in order to prepare the DNA for cell-free reactions. We used the ZYMO kit and followed the MaxiPrep procedure.

Nanodrop Results of C4HSL Induced RhlR Plasmid (Maxiprep #1)

	A	B	C
1		EB	RhlR
2	Ratio (260/280)	1.13	1.85
3	Concentration (ng/μL)	-1.3	79.9

6/17/2019 - 6/21/2019

Monday

We made an overnight today for another Maxiprep. It was also taken from glycerol stick #7 (A.E.), but this time was made with200 mL of LB broth and 200 μL of chloramphenicol. This overnight was put in at 37o C overnight.Then LabPats made overnights of 6 different stocks of 3OC12 in pLas plasmid (the sfGFP 2017 part) in order to find the one that fluoresced to be used for the characterization of an existing part. There were pLas B5, pLas from 6/29/17, pLsr D5, pLsr F2, pLsr E0, and pLsr D2. We also made overnights of their DMSO and a blank that had 3μL of chloramphenicol per 3 mL of LB. There were 13 tubes total which were put in the incubator at 37o C overnight.

Tuesday

We checked the overnights and they did not glow because pLsr does not glow (wrong plasmid) and either of the pLas we did use were not the one that worked in 2017. Therefore we made more overnights (3μL chlor. per 3mL LB) and 3μL of 3OC12 in pLas plasmid (sfGFP) along with their corresponding DMSO and a blank (15 tubes total).

1. pLas_sfGFP miniprep TD#1
2. pLas_sfGFP miniprep TD#2
3. pLas_sfGFP miniprep TD#3
4. pLas_sfGFP #6 JD
5. pLas_sfGFP #3 HJ
6. pLas_sfGFP #5 JD
7. pLas_sfGFP #4 HJ

Another maxiprep of RhlR was done but with some modifications in order to try and increase the DNA amount. The team used 200mL of RhlR instead of 150mL, heated the EB to 50o C, used 200 μL of EB instead of 400L, and let the EB sit for 10 minutes prior to spinning down.

Nanodrop Results of Maxiprep #2

	A	B	C
1		EB	C4HSL
2	ratio 260/280	0.88	1.84
3	concentration (ng/μL)	0.5	79.7

Wednesday

One of the overnights fluoresced pLas_sfGFP_JD #5. The LabPats made triplicates with this plasmid along with the corresponding DMSO and a blank. Then we made an ethanol precipitation of the two RhlR MaxiPreps from 6/14 and 6/18 in order to increase the concentration of DNA.

MaxiPrep Results after Ethanol Precipitation

	A	B	C
1		ratio 260/280	concentration ng/mL
2	#1 (6/18)	1.83	87.1
3	#2 (6/14)	1.87	243.1
4	EB	-0.4	0.62

For measurements, wells E-G 5-11 were the dilution for sfGFP (green to white) and wells B-D 5-11 were the dilution for fluorescence (blue to white). The C4-HSL dilution was 20 fold (.5μL of C4HSL and 9.5 μL of DMSO).

Wells B2-B4

negative control (red)

10μL premix

9μL extract

6μL water

Wells C2-C4

positive control (purple)

10μL premix

9μL extract

3μL DNA (RhlR)

3μL water

Wells D2-D4

DMSO w/o polymerase (light green)

10μL premix

9μL extract

4.5μL DNA (RhlR)

0.5μL DMSO

1μL water

Wells E2-E4

C4HSL w/o polymerase (light blue)

10μL premix

9μL extract

4.5μL DNA (RhlR)

0.5μL C4HSL

1μL water

Wells F2-F4

DMSO w/polymerase (E. coli RNA Polymerase Holoenzyme S M0551S from New England Biolabs) (yellow)

10μL premix

9μL extract

4.5μL DNA (RhlR)

0.5μL DMSO

1μL polymerase

Wells G2-G4

C4HSL w/polymerase (E. coli RNA Polymerase Holoenzyme S M0551S from New England Biolabs) (orange)

10μL premix

9μL extract

4.5μL DNA (RhlR)

0.5μL C4HSL

1μL polymerase

The Promega Kit (S30 T7 high-Yield Protein) Expression System was used for these cell-free reactions. The master mix protocol was followed (see above) and put in a 96 well plate. The plate was covered with a non breathable membrane. MaxiPrep #1 from 6/18 was used. The plate (REF L1110) was in the plate reader overnight.

Settings for fluorescence in plate reader: 480-510λ, bottom read every 10 minutes for 12 hours at 30oC, 5 seconds shaken, black well w/ clear bottom

Thursday

Today the LabPats measured the 2017 iGEM part. This began with a dilution of the cultures (50μL of LB and 50μL of culture). A1-A3 were samples of 30C12 JD #5, A4-A6 were samples of DMSO, and A7 was the blank in a 96 well plate.Plate Reader
Settings for absorbance: 600 OD, top read, purple adapter for bottom, 10 seconds shaken, clear wells, 22.7oC Settings for fluorescence: 480-510λ, bottom read, 5 seconds shaken, black well w/ clear bottom, 22.7oC

Friday

The LabPats prepared for their meetup on Saturday.

06/24/2019 - 06/28/2019

Monday

Two bottles of LB were made. It was in the autoclave for 15 minutes with the flashing sign/liquid cycle. We made sure that the bottles were not too tightly sealed and had autoclave tape on them. The bottles were labeled LB broth iGEM 6/24/19.

Tuesday

An overnight of RhlR for a new MaxiPrep was made. A sterile bottle was used since we did not have sterile flasks. The solution included 200mL of LB Broth, 200μL of chloramphenicol, and RhlR stock #7 A.E. It was placed in the incubator at 37oC overnight (we made sure the cap wasn't too tight)

Wednesday

The MaxiPrep of the overnight from 6/25 was completed.

Nanodrop results from MaxiPrep of RhlR

	A	B	C
1		ratio 260/280	concentration ng/ml
2	RhlR	1.89	3381.5
3	EB	1.48	-0.6

The cell-free reactions were then set up using the DNA from the MaxiPrep. The same protocol was followed as on 6/19 but with 0.6μL of DNA for each reaction (DNA Calculation 0.6μL * 3382 = 2000ng so we want 2μg of DNA). Therefore, the adjusted reactions were as follows:

B2-B4, negative control
C2-C4, positive control
D2-D4, DMSO w/o polymerase, 0.6μL DNA, 4.9 μL H2O

E2-E4, C4HSL w/o polymerase, 0.6μL DNA, 4.9 μL H2O

F2-F4, DMSO, 1μL polymerase, 0.6μL DNA, 3.9μL H2O

G2-G4, C4HSL, 1μL polymerase, 0.6μL DNA, 3.9μL H2O

Plate reader settings: endpoint reading for fluorescence, read from the bottom, 480-510λ, sensitivity 19, autoread 600, temperature 30o C.

Thursday

The LabPats did a data analysis of the measurements from the plate reader. The data showed that the RhlR circuit #2 did not work. So we decided to test with the S30 E.coli (Extract for circular plasmid) native kit. This is a Promega Kit (cat. # L1020). This cell free reaction has more steps to the protocol than previous 25μL reactions, including complete amino acid for all the reactions (which is a mixture of amino acid without methionine and amino acid without leucine) and synthesis and assays of luciferase for the positive control.

Reaction A -

No DNA: wells B2-B4

DMSO: wells D2-D4

10μL Premix- 7.5μL Extract- 2.5μL complete amino acid (RhlR)5μL- nuclease free water

Reaction B -

pY71: wells C2-C4

10μL Premix- 7.5μL Extract- 3μL DNA (pY71-sfGFP)- 1μL T7 Polymerase- 2.5μL amino acid

Reaction C -
10μL Premix- 7.5μL- 0.6μL DNA- 0.5μL DMSO- 2.5μL amino acid- nuclease free water

Reaction D -

C4-HSL: wells E2-E4

originally 10μL Premix

prior to

7.5μL Extract- 0.6μL DNA (RhlR)- 0.5μL C4-HSL- 2.5μL amino acid- 3.9μL nuclease free water- 1μL nuclease free water- 3.9μL

Blank -

wells F2-F4

There is nothing in these wells because they weresupposed to be luciferase, but we did not incubate (step 3 in protocol) starting reactions B-D.

Friday

Today's focus was on redoing the luciferase control because it was not measured on Thursday as planned. There were two reactions: the luciferase and no DNA. The luciferase required incubation for an hour at 37o C, an ice bath for 5 minutes, and a dilution series before being pipetted into a 96 well plate. It was originally done in wells G1-8 and H1-12 (G1-G4 had the stock solution, G5-G8 had the 1st dilution, H1-H4 had the 2nd dilution, and H9-H12 had no DNA). Then the experiment was redone. now with B2-B5 having the stock solution, B6-B9 had the 2nd dilution, C2-C5 had the 3rd dilution, C6-C9 had the 4th dilution, and D2-D5 had no DNA.

Luciferase Reaction
2μL pBESTluc DNA2.5μL complete amino acid mix- 10μL Premix- 7.5μL Extract- 3μL Nuclease free water

No DNA Reaction 10μL Premix- 7.5μL Extract- 2.5μL Amino acid- 5μL Nuclease free water

7/1/2019 - 7/5/2019

Monday

The LabPats lyophilized three reactions: one with no DNA, one with pY71-sfGFP added before being rehydrated and one with pY71-sfGFP added after being rehydrated (it was resuspended with DNA and water). The purpose of this was to see if functionality was improved with DNA being added before or after. For each (no DNA, pY71 before, and pY71 after), we did a 25μL reaction and four 5μL reactions. These reactions froze in the -80o freezer for 10 minutes and then in liquid nitrogen for 1 minute. After this, the caps were removed and put in the lyophilizer overnight in order to remove the liquid.

No DNA reaction 10μL Premix- 9μL Extract- 6μL Water

pY71-sfGFP DNA after- 10μL Premix- 9μL Extract- 6μL Water- (Resuspended with DNA and water)

pY71-sfGFP with DNA before 10μL Premix- 9μL Extract- 3μL pY71-sfGFP- 3μL Water

Tuesday

The reactions from Monday (7/1) were rehydrated and run through the plate reader, showing that the lyophilization was successful. Reactions 1 and 2 were rehydrated with the same amount of water as the reaction (so the 25μL rxn was rehydrated with 25μL of water and 5μL water with 5μL reactions). Reaction 3 had both water and pY71-sfGFP DNA that was rehydrated in 3μL DNA and 22μL water. We took pictures of the plate for visual proof of fluorescence.Since our cell free reactions had not been successful, LabPats began troubleshooting. We redid our cell free reactions in both T7 and S30 kits, but with the native control pAME 215. pAME 215 is a strong, modified native control which we used as a replacement for pY71-sfGFP since it didn’t work previously in the S30. We did these in 5 μL reactions with no DNA, pAME 215, or pY71-sfGFP in both kits.

T7 No DNA (B2-5 and D2-5)

10μL Premix
9μL Extract
6μL Water

1μL Water

S30 No DNA (C2-5)
10μL Premix
7.5μL Extract
2.5μL Complete amino acid mixture

5μL Water

T7 pAME 215 (B6-9)

10μL Premix

9μL Extract
5μL Native control

1μL Water

S30 pAME 215 (C6-9)

10μL Premix

7.5μL Extract

5μL pAME 215
2.5μL Complete amino acid

T7 pY71-sfGFP (E2-5)

10μL Premix
9μL Extract
5μL pY71-sfGFP

Then we did an experiment to test if DMSO was having an effect on the cell free reactions. We used the T7 kit for this and had reactions of no DNA, pY71-sfGFP + DMSO, and just pY71-sfGFP.

No DNA

10μL Premix

9μL Extract

6μL Water

pY71 + DMSO

10μL Premix

9μl Extract
3μL pY71-sfGFP

0.5μL DMSO

2.5μL Water

pY71
10μL Premix

9μL Extract

3μL pY71-sfGFP

3μL Water

Wednesday

Troubleshooting continued today with DNA titrations. We had been doing our cell free systems with a final concentration of 80ng/μL, and we were advised that we may be overloading the system with this amount of DNA. With this is mind, we did our titrations with 20, 40, 60, and 80ng/μL of DNA along with corresponding DMSO. Then we put the reactions in the plate reader overnight.

Reaction A (No DNA) B2-B5

10μL Premix
9μL Extract

6μL Water

Reaction B (+ control pAME215) B7-B10

10μL Premix
9μL Extract

5μL DNA

1μL Water

Reaction C (80ng/ μL with DMSO) C7-C10

10μL Premix
9μL Extract

0.6μL DNA (RhlR)

0.5μL DMSO

Reaction D (80ng/μL)

C2-C5

10μL Premix
9μL Extract
0.6μL DNA (RhlR)

0.5μL C4HSL (5mM)

4.9μL Water

9μL Extract

5μL DNA

1μL Water

Reaction E (60ng/μL with DMSO)

D7-D10

10μl Premix
9μL Extract
4.4μL DNA (RhlR)

0.5μL DMSO
1.1μL Water

9μL Extract
0.6μL DNA (RhlR)

0.5μL DMSO

Reaction F (60ng/μL)

D2-D5

10μL Premix
9μL Extract
4.4μL DNA (RhlR)

0.5μL C4HSL(mM)

1.1μL Water

Reaction I (20ng/μL with DMSO)

F7-F10

10μL Premix
9μL Extract
1.5μL DNA (RhlR)

0.5μL DMSO

4μL Water

Reaction G (40ng/μL with DMSO)

E7-E10
10μL Premix
9μL Extract

5μL DNA (RhlR)

0.5μL DMSO

0.5μL Water

Reaction J (20ng/μL)

F2-F5

10μL Premix
9μL Extract
1.5μL DNA (RhlR)

0.5μL C4HSL (5mM)

4μL Water

Reaction H (40ng/μL)

E2-E5

10μl Premix
9μL Extract
5μL DNA (RhlR)

0.5μL C4HSL (5mM)

0.5μL Water

Thursday

LabPats were off for 4th of July.

Friday

LabPats were off for the 5th of July

7/8/2019 - 7/12/2019

Monday

We received new plasmids from RX. They are pY71-bFMO, C4HSL-bFMO, and pET11a-ChrB. We followed the miniprep protocol to miniprep the pY71-bFMO which were labeled pY-bFMO 1 and pY-bFMO 2. pY71-bFMO has resistance to the antibiotic kanamycin.

MiniPrep of pY-71bFMO

	A	B	C
1		concentration	ratio
2	miniprep #1	53.3	1.83
3	miniprep #2	63.8	1.85
4	EB	0.0	1.04

Then we used miniprep #2 for the transformation. The plates were labeled pY71-bFMO BL21-DE3 1X and pY71-bFMO BL21-DE3 9X. BL21-DE3 are competent E. coli cells. The plates were incubated overnight.We also followed the maxiprep protocol with both the old and new kits in order to maxiprep all three of the plasmids, and we made glycerol stocks of them. The maxiprep for pY71-bFMO was spilled during the procedure so we could not get data from it.

Maxiprep for new plasmids

	A	B	C
1		Concentration ng/μL	ratio 260/280
2	pY-bFMO #1	no data	no data
3	pY-bFMO #2	no data	no data
4	C4HSL bFMO #1	52.0	2.00
5	C4HSL bFMO #2	37.1	1.81
6	pET11a-ChrB #1	50.2	1.95
7	pET11a-ChrB #2	60.6	1.93
8	EB	-0.3	0.69

We made 200mL overnights of each plasmid with 200μL of the corresponding resistance. For pY71-bFMO that is Kanamycin, for pET11a-ChrB that is Ampicillin, and chloramphenicol for C4HSL-bFMO. We induced C4HSL-bFMO sterile with 5μL of C4HSL and also made 3 overnights of C4HSL-bFMO using the glycerol stock that was made earlier and 3μl of C4HSL, 3 other overnights with 3μl of DMSO and 1 blank consisting of 3mL of LB Broth.

Tuesday

LabPats followed the maxiprep protocol for each of the 200μL overnights from yesterday.

Maxiprep for overnights of bFMO and ChrB

	A	B	C
1		concentration ng/μL	ratio 260/280
2	pY71-bFMO	184.7	1.9
3	pY71-C4HSL	709,1	1.87
4	pET11a-ChrB	910.4	1.88
5	EB	-0.8	0.76

Then we redid the transformations since the ones from yesterday were not successful. The overnights from last night did not express bFMO so we made new overnights but this time with tryptophan added. We did not use straight tryptophan but a recipe of it dissolved in water. The recipe was 200μL of milli-q water and 1g of tryptophan which is 5mg/μL. For pY71-bFMO, there was one blank with 3mL of LB broth and 3μL Kan, 3 tubes of pY71-bFMO with 3mL of LB broth and 3μL Kan, and 3 tubes of pY71-bFMO with tryptophan added. These last tubes had 2.4mL of LB, 600μL of tryptophan, and 3μL Kan. The same was for the C4HSL-bFMO except the antibiotic was chloramphenicol.

Wednesday

We checked the overnights and saw that some of the pY71-bFMO with tryptophan had expression of bFMO. However, none of the C4HSL-bFMO tubes expressed bFMO. We looked at the transformation and there were tiny colonies and a film on the plates so we redid the transformation using the same protocol from 7/9/19 except using 50 Kam plates instead of 25 Kam. We streaked three plates for each transformation (at 1X and 9X concentration).Then the LabPats did cell free reactions with pY71-bFMO since cell free reactions with C4HSL were not working. This DNA was tested alone and then with tryptophan, with DMSO, and with both in quadruplicates. The plates were put in the plate reader overnight with the absorbance set at 620nm and an endpoint reading for the positive control (pY71-sfGFP) was taken the next morning.

RXN A (No DNA)

Wells B2-B5

10μL Premix
9μL Extract
6μL Water

RXN B (pY71-sfGFP)

Wells B6-B9

10μL Premix
9μL Extract
3μL Water

3μL pY71-sfGFP

RXN C (pY71-bFMO)

Wells C2-C5

10μL Premix
9μL Extract

2.8μL Water

3.2μL pY71-bFMO

RXN D (pY71-bFMO +Trp)

Wells C6-C9
10μL Premix
9μL Extract

3.2μL pY71-bFMO

2.3μL Trp
0.5μL Water

RXN E (pY71-bFMO +DMSO)

Wells D2-D5

10μL Premix

9μL Extract

3.2μL pY71-bFMO

0.5μL DMSO

2.3μL Water

RXN F (pY71-bFMO +both)

Wells D6-D9

10μL Premix

9μL Extract

3.2μL pY71-bFMO

0.5μL DMSO

2.3μL Trp

Thursday

The transformations from 7/10 were successful so we made overnights from them. The results of the cell free system showed visual blue at the bottom of some of the wells and the data analysis showed slightly higher absorbance readings for pY71- bFMO by itself, with DMSO and with Tryptophan compared to No DNA and pY71-bFMO with both DMSO and Tryptophan. We have been using tryptophan in the past experiments because tryptophan is converted into indole by E.coli which reacts with bFMO to express an indigo color. With us putting tryptophan into the reactions, it was expected that more indigo would be expressed. With positive results from the last overnights with Trp, we decided to make overnights with tryptophan, IPTG (which is supposed to induce T7 polymerase allowing for greater production from our plasmid), both Trp and IPTG, and neither. These overnights were quadruplicates made from 4 different colonies, resulting in 17 overnights including the blank.

Cells as seen in the table means 20μL LB and one of the colonies (1, 2, 3, or 4) that was then aliquoted into four 5μL tubes in order to be put into A, B, C, and D overnights (see left)

A = with IPTG
B = neither

C = with IPTG and Trp

D = with Trp

Recipe for overnights with and without Trp and IPTG

	A	B	C	D	E	F
1	Label	Cells	Trp (final concentration1mg/mL)	1M IPTG (final concentration0.5mM)	LB	50 KAN
2	A	5μL	-	1.5μL	3mL	3μL
3	B	5μL	-	-	3mL	3μL
4	C	5μL	600μL	1.5μL	3mL	3μL
5	D	5μL	600μL	-	3mL	3μL
6	Blank

Friday

LabPats checked the overnights. Group D tubes (with tryptophan) expressed a lot of indigo and were very dark. This confirmed that bFMO works in cells. We also saw that IPTG seemed to kill the reaction since tubes with IPTG in them weren't as dark (both with and without Tryptophan). Colony 4 did not grow in reactions A and C. We made glycerol stocks labeled B1- B4 since B was the label for overnights without IPTG and Trp.In order to measure the amount of indigo expressed by bFMO, we must extract the indigo using DMSO and spin the pellet down since indigo and cells have the same absorbance, and therefore simply putting the overnights in the plate reader would not give us the amounts of indigo. While we knew how to do the extraction protocol, we were not sure of the correct amount of overnight or DMSO needed for our supernatant to not be too diluted but also remove as much of the indigo from the pellet as possible. This led to a lot of trial and error.

We began by placing 1000μL of overnight into a 1.5μL tube except for those with colony 4 which did not have much growth if at all. These were spun down in the centrifuge at 13,400 rpm xg for 5 minutes. We took out the supernatant, careful not to remove the pellet with it. Then we resuspended with 500μL of DMSO. When this did not yield the results we wanted, we experimented with extraction volumes.Samples A2 and A3. 10μL, spin, add 10μL DMSO, spin, add 20μL, spin, add 60μL, spin, remove into separate tube, add 200μL, spin, remove and combine with supernatant in separate tube, add 200μL. For A2, add another 20μL, spin, remove to separate tube, add 20μL, spin, remove to separate tube, add 20μL, and remove to separate tube.Samples D2 and D3: 500μL, spin, remove to separate tube, add 500μL, remove to separate tube, add 1mL, spin, remove to separate tube, add 1mL, spin, remove to separate tube, add 1mL, spin, remove to separate tube, and 20μL and spin down.Samples D1 and A1: 500μL, spin, For A1: remove to separate tube, add 500μL, For D1: remove to separate tube, add 1mL, spin down, remove to separate tube, add 1mL, spin, remove to separate tube, add 500μL, spin down.Samples B1-B3 and C1-C3: 500μL, spin, separate into a new tube, add 500μL, spin, separate, add 500μL. Then, we ran a few of these on the plate reader at absorbance at 620nm.

bFMO Extraction

Table1

	A	B	C
1	Plate and Volume	DMSO Abs. (a.u)	pY71-bFMO Abs. (a.u)
2	V-bottomed,5ul	1.009	0.992
3	V-bottomed, 20 μL	1.293	1.413
4	Flat, 100 μL	0.039	1.189
5	Flat, 25 μL	0.036	0.114
6	Flat, 50 μL	0.033	0.451

Table2

	A	B	C	D
1	Flat, 100 μL	Well #1 Abs. (a.u)	Well #2 Abs. (a.u)	Well #3Abs. (a.u)
2	pY71-bFMO + IPTG	0.432	0.661	0.564
3	pY71-bFMO	0.691	0.597	0.686
4	pY71-bFMO + Trp + IPTG	0.912	0.296	0.458
5	pY71-bFMO + Trp	1.153	1.116	1.017

Table3

	A	B
1	Flat, 100 μL	Well #1
2	Empty Well	0.135
3	Water	1.016

7/15/19 - 7/19/2019

Monday

Monday morning consisted of preparing for our lab meeting presentation. In the afternoon, we redid overnights of pY71-bFMO in triplicates with 3mL of LB and 3μL Kanamycin and the glycerol stock B1 from 7/12/19.

RXN A: pY71-bFMO
RXN B: pY71-bFMO + 600μL TrpRXN C: pY71-bFMO + 1.5μL IPTG
RXN D: pY71-bFMO + 600μL Trp + 1.5μL IPTG RXN E: 100μL BL21 DE3 competent cells Blank: 3mL of LB

Tuesday

Today LabPats redid the bFMO extraction, this time taking 50μL of each overnight, spinning it down at 13,400g for 5 minutes, discarding the supernatant, adding 500μL of DMSO, spinning it down again, and removing the supernatant to a different tube to be measured. Then, to test which amount gave the most accurate readings, we aliquoted 5μL, 10μL and 25μL into a 384 well plate and then run in the plate reader. The bL21-DE3 cells were placed directly from the overnight with no extraction protocol followed. This gave us inaccurate results so we redid the reading with BL21-DE3 that had been through extraction.

RXN A: Blue

RXN B: Red

RXN C: Yellow

RXN D: Green

RXN E: Black

RXN F: Purple
C2, E2 and G2 were not measured with RXN D because the extracted supernatant was accidentally thrown into biohaz Rows B and C were 5μL reactions, Rows D and E were 10μL reactions, and Rows F and G were 25μL reactions

Absorption Data from bFMO Extraction #2

Wednesday

We autoclaved empty flasks and bottles using the dry cycle on the autoclave. Then we did an overnight of the PLas_sfGFP circuit JD #5. It included 200mL of LB and 200μL of chloramphenicol.We also did a rehydration and transformation of TetR, part: BBa_K876004 (pTET-RBS-GFP-T-pLac-TeTq) which came from the iGEM Distribution Kit in order to check off the measurement criteria from the bronze medal. For this transformation we used 2018 plate #2 Well 15N. We punched a hole through the desired well using a pipette tip, pipetted 10μL of dH2O up and down, and resupspended the DNA which turned red. Then we transformed the DNA and placed the 9μL that were left into a tube labeled pTET. LabPats also redid the pY71-bFMO Cell Free System reactions. They were redone in triplicates and ran overnight on the plate reader at an absorbance of 620nm. The DNA was at a concentration of 180ng/μL

RXN A (No DNA)

Wells B2-B4

10μL Premix
9μL Extract
6μL Water

RXN D (pY71-bFMO +Trp)

Wells C2-C4
10μL Premix
9μL Extract3.2μL pY71-bFMO 2.3μL Trp
0.5μL Water

RXN B (pY71-sfGFP)

Wells B5-B7

10μL Premix
9μL Extract
3μL pY71-sfGFP

3μL Water

RXN C (pY71-bFMO)

Wells B8-B10

10μL Premix
9μL Extract
3.2μL pY71-bFMO

2.8μL Water

RXN F (pY71-bFMO +both)

Wells D6-D9

10μL Premix 9μL Extract

3.2μL pY71-bFMO

0.5μL DMSO

2.3μL Trp

RXN E (pY71-bFMO +DMSO)

Wells C5-C7

10μL Premix

9μL Extract

3.2μL pY71-bFMO

0.5μL DMSO2.3μL Water

We also lyophilized which meant making the same 25μL reactions with DNA as above and putting them in 0.6mL (600μL) tubes. Next, we froze the reactions in liquid nitrogen for 1 minute. Then we removed the caps and put the tubes in the lyophilizer overnight to remove the liquid.

The LabPats also made overnights for the 3OC12 Dose Response Curve. Each overnight had 3mL of LB, 3μL of 3OC12 stock (diluted with DMSO), and pLas_sfGFP JD #5. The 3OC12 stocks began with 20μL and was diluted into 10μL of DMSO down the line of tubes so that each had a total of 10μL but half as concentrated with 3OC12 as the previous tube. Then the 10μL were aliquoted into the triplicates so each had 3μL of the 3OC12. They were then put in the incubator at 37oC.

Thursday

Rehydrating in pr
Maxiprep
Transformation used 2018 kit
Rehydrated Cell free systems because we forgot to put on membrane, put in incubator for overnight

Friday

Got endpoint reading for plate
Made LB broth
5μL standard curve
Bioinformatics session with Vanessa Varaljay.

7/22/2019 - 2/26/2019

Monday

LabPats redid the 5μL standard curve because the wrong plate was used for Friday's
The overnights for 3OC12 dose response curve were set up. The one from 7/17 was not diluted properly so we needed to redo them.

Tuesday

RH members went over to RX to work with running gels.LabPats analyzed data. Only T7-mutated with and without Trp worked (turned blue). No GFPa1 circuit fluorescence was detected. The transformation of ChrO-bFMO (on a 20ng Kan plate) and ChrP-GFPa1 worked.A bFMO extraction was also done. 50μL of the overnight was added to 750μL of DMSO.
mg/mL DMSO * 750μL DMSO/50μL culture. Multiply the raw data by 15 and convert using the indigo standard curve (conversion factor 5.30E-5)

Wednesday

LabPats spent the day at UES working on wiki content, data analysis consisting mostly of converting our readings to MEFL/particle, and our presentation for Dr. Rajesh Naik. At 1:00pm RX members came over to RH to get photos of the entire team doing labwork with the base photographer. Then RH went back to UES to continue working.

Thursday

Cell free reactions w/bFMO were done.
Plate Setup was 25μL reactions in a 384 well plate B2-B4: T7 mutated bFMO + Trp
B5-B7: T7 mutated bFMO
B8-B10: ChrP bFMO + Trp
B11-B13 ChrP bFMO

B14-B16 DMSO only

Lyophilization of cell free reactions was also done.

Friday

Labpats rehydrated the reactions for Thursday.
A lot of time was spent talking with our mentor, Amy, about was to complete medal criteria of measurement (bronze), validation (silver) and improvement of an existing part (gold).

7/29/2019 - 8/1/2019

Monday

LB broth was made, The ChrP-mRFP was transformed and C4-HSL Dose response curves were done and put in the plate reader(Amy glycerol stock #7)
Overnights of ChrP-bFMO, chrP-GFPa1, ChrP-bFMO + Trp, pY71-bFMO, and pY71-sfGFP were made and put in the incubator. LabPats tested the paper sensor with fluorescein. After seeing success, cell free tests on paper were run with pY71-sfGFP and bFMO (w/DNA, Trp, indole made indole)

Tuesday

Maxipreps
Cell free reaction

Wednesday

One member of RH took the cell free data off the plate reader and graphed it. Then the team met at UES to practice the presentation for Dr. Naik.

Thursday

Presented to Dr. Naik
Ran cell free reactions in triplicates. One reaction had No DNA + DMSO and the other had pY71-bFMO + DMSO Concluded DMSO had no effect on the reaction

8/5/2019 - 8/9/2019

Monday

Checked different wavelengths for pY71-bFMO absorbance measurements (470 nm vs 620 nm) using the DMSO testing from 8/1 and using ChrP-bFMO colonies in water and there was no difference in measurements. Realized that our T7 promoter had an illegal XbaI site so we began a mutagenesis to remove it.
Made more 50 Kan plates, followed protocol

Began performing single point mutagenesis to mutate an illegal XbaI cut site in between our T7 promoter and RBS region in our pY71-sfGFP

We also plated the blank on a non-resistance (NR) plate.
Made a 200 mL overnight of the PET11a-chrB Sample 1 Colony 1 to maxiprep (Ampicillin resistance)

Tuesday

Transformations were mostly unsuccessful. Blank plated on NR plate only had a slight film. The 5 uL, 20 ng/μL reaction did have a few colonies. So we made 7 overnights (6 colonies + 1 Blank) with 3 mL of LB, and 3 μL of 50 ng Kanamycin. Maxiprep was unsuccessful because there was some confusion over the last two steps that led to a contaminated sample. So we redid a 200 mL overnight to repeat the maxiprep again tomorrow. We concluded that the older DH5α cells were used so we used newer DH5α cells to repeat the mutagenesis of the 20 ng/μL reaction. We also began a mutagenesis for the T7 promoter in our pY71-bFMOplasmid using the same protocol with minor revisions.

Master Mix for 7 reactions (4 pY71-bFMO, 1 pY71-sfGFP, 1 Blank, 1 extra)

5 iProof Buffer- 35μL

DNA

T7mut-F- 3.5μL (10x dilution of primers- 1 μL of each primer to 9 μL of water)

T7mut-R- 3.5μL
DNTP- 3.5μL
Water- 122.5 μL
iProof polymerase- 3.5μL
Total: 171.5/7=24.5 μL per reaction

pY71-sfGFP (20 ng/μL)
dilute 1 μL of DNA to 9 μL of water

Add 0.5 μL to corresponding reaction

pY71-bFMO - dilute 1 μL of DNA to 9 μL of water- 246.54 ng/μL is about 250 ng/μL 5 ng/μL= 1:50 (49 μL) of water
10 ng/μL= 1:25 (24 μL) of water
20 ng/μL= 1:12.5 (11.5 μL) of water 50 ng/μL= 1: 5 (4 μL) of water
Add 0.5 μL of each diluted DNA sample to corresponding reactionPCR Revisions:

For pY71-sfGFP: 68 °C for 75 seconds

For pY71-bFOMO 68 °C for 100 seconds

Left transformations overnight. Autoclaved some flasks, bottles, and μL tubes.Sent pY71-sfGFP, pY71-bFMO, ChrP-GFPa1, ChrP-bFMO for sequencing Primers (10x dilution needed):

seq_pY71_F

seq_pY71_R

pY71-sfGFP F- BGC225

pY71-sfGFP R- BGC226

pY71-bFMO F- BGC227

pY71-bFMO R- BGC228

ChrP-GFPa1 F- BGC229

ChrP-GFPa1 R- BGC230

ChrP-bFMO F- BGC231

ChrP-bFMO R- BGC232

Wednesday

The maxi prepped sequences for PET11a-ChrB were confirmed.

Nanodrop for Maxiprep of Pet11A-ChrB

	A	B	C
1		Concentration (ng/μL)	Ratio 260/280
2	PET11a	360.6	1.9
3	EB	-0.7	0.87

LabPats mini prepped pY71-sfGFP (5μL, 20ng/μL) overnights using miniprep kit. Glycerol stocks of this were made and labeled GS1, GS2... GS6 sfGFP. They are in the iGEM 2017 box.T7-mut-pY71-sGFP

Miniprep Nanodrop Results

	A	B	C
1		Concentration (ng/μL)	ratio 260/280
2	EB #1	-0.8	169.11
3	EB #2	-0.1	0.75
4	T7-mut-sfGFP col. 1	143.0	1.88
5	T7-mut-sfGFP col. 2	82.4	1.91
6	T7-mut-sfGFP col. 3	83.8	1.90
7	T7-mut-sfGFP col. 4	176.7	1.65
8	T7-mut-sfGFP col. 5	89.3	1.90
9	T7-mut-sfGFP col. 6	87.9	1.92
10	EB #3	0.2	2.31

LabPats also did sequencing. The set-up was 8μL of DNA and 4μL of 10X primers.

T7 mut-sfGFP #1 F BGC233 R BGC 234

T7 mut-sfGFP #2 F BGC235 R BGC 236

T7 mut-sfGFP #3 F BGC237 R BGC 238

T7 mut-sfGFP #4 F BGC239 R BGC 240 (this sequence was confirmed!)

T7 mut-sfGFP #5 F BGC241 R BGC 242

T7 mut-sfGFP #6 F BGC243 R BGC 244

Finally, overnights of pY71-bFMO (T7-mut) were made with 3mL of LB and 3μL of 50 Kan.

Thursday

Glycerol stocks of T7 mut-pY71-bFMO were made. They are labeled as GS bFMO 1... GS bFMO 10. 100μL of each overnight was put into a 60% glycerol stock.Following the protocol from 8/7/2019, the overnights were mini prepped and nano dropped. (ng, μL) in the segment # column is for mutagenesis cone and transformation amount. For #1 and #9 DNA was diluted.

Nanodrop Results of Miniprepped Overnights

	A	B	C	D
1	Segment #	Concentration (ng/μL)	Ratio 260/280
2	EB #1	0.3	0.90
3	BGC 245 T7- mut-bFMO #1(5ng, 1μL)	173.5	1.75	*sequenced confirmed*
4	BGC 246 T7- mut-bFMO #3(5ng, 5μL)	156.1	1.86
5	BGC 247 T7- mut-bFMO #2(5ng, 5μL)	138.1	1.86
6	BGC 248 T7- mut-bFMO #4(10ng, 1μL)	130.6	1.86
7	BGC 249 T7- mut-bFMO #5(10ng, 1μL)	117.7	1.85
8	BGC 250 T7- mut-bFMO #6(10ng, 5μL)	128.6	1.85
9	BGC 251 T7- mut-bFMO #7(10ng/5μL	121.4	1.86
10	BGC 252 T7- mut-bFMO #8(10ng/1μL)	158.3	1.79
11	BGC 253 T7- mut-bFMO #9(20ng, 5μL)	187.4	1.80
12	BGC 254 T7- mut-bFMO #10(20ng, 5μL)	102.5	1.86
13	BGC 255 T7- mut-sfGFP #2(20ng, 5μL)	137.3	1.85
14	EB #2	0.4	0.52

LB broth and a sleeve of 50Kan plates were also made.

Friday

LabPats began making the indigo standard curve. 7.28mg of indigo was put into 728μL of DMSO so there was a 0.01ng/μL concentration. 60μL of indigo was put into the 1st PCR tube. Each successive tube had 30μL DMSO in them. 30μL of indigo from tube 1 was pipetted into tube 2, 30μL of tube 2 was pipetted into tube 3, etc until liquid became clear (approximately 16 tubes). This was run on the plate reader at an absorbance at 620nm in a 384 well plate. The results were inconsistent so the standard curve was repeated. The 17th well in the plate was a blank of pure DMSO.

8/12/2019 - 8/16/2019

Monday

An overnight of T7-mut-pY71-sfGFP #4 was made to maxiprep. It consisted of 200mL of LB and 200μL of 50Kan. Overnights of pY71-sfGFP and T7-mut-pY71-sfGFP #4 were also made for measurement. These had 3mL of LB, 3μL of 50Kan, and were inoculated with G5 J7-mut-sfGFP #4 or pY71-sfGFP maxiprep.Received chrP-bFMO and chrP-GFPa1 minipreps and NEB5 plate with potentially an RBS in it. There were 4 minipreps each with an approximately 200ng/μL concentration for each. These were not nanodropped. Primers bFMO425-443, bFMO932- 950, and C4-HSL-bFMO seg Rev were received. Sequencing set-up was 8μL of DNA and 4μL of 10X primers.

pY71-bFMO (redo) BGC256

ChrP-bFMO #1 BGC257 (443 primer)

ChrP-bFMO #1 BGC258 (950 primer)

ChrP-bFMO #1 BGC259 (F seg-pY71- F primer)

ChrP-bFMO #2 BGC260 (443)

ChrP-bFMO #2 BGC261 (950)

ChrP-bFMO #2 BGC262 (F)

ChrP-bFMO #3 BGC263 (443)

ChrP-bFMO #3 BGC264 (950)

ChrP-bFMO #3 BGC265 (F)

ChrP-bFMO #4 BGC266 (443)

ChrP-bFMO #4 BGC267 (950)

ChrP-bFMO #4 BGC268 (F)

ChrP-GFPa1 #1 BGC269 (F)

ChrP-GFPa1 #2 BGC270 (F)

ChrP-GFPa1 #3 BGC271 (F)

ChrP-GFPa1 #4 BGC272 (F)

Also sequenced 3OC12 and RhlR circuits (10X dilution of maxipreps)

pLas-sfGFP- BGC273- AME1012

pLas-sfGFP- BGC274- AME1013

RhlR- BGC275- AME1012

RhlR- BGC276- AME1013

Made 4 overnights in triplicates of pY71-bFMO (NEB5) from a glycerol stock and plate from RX to check for mutation because there was a point mutation in our pY71-bFMO that caused an amino acid change. The overnights consisted of 3mL of LB and 3μL of 50Kan. 3 overnights were from the pY71-bFMO glycerol stock #1 and 9 overnights were made from the plate (3 from sample 1, 3 from sample 2, and 3 from sample 3).

Tuesday

Overnights of the pY71-sfGFP did not grow anything so LabPats redid them using a colony from a 9X pY71-sfGFP (DH5∝) plate from 7/22/19. 20μL of LB + colony meant that each overnight was 5μL. 4 overnights of T7-mut-sfGFP #4 were made from glycerol stock #4 (also in DH5∝)T7-mut-sfGFP #4 was maxiprepped.

Nanodrop Results of Maxiprepped T7-mut-sfGFP #4

	A	B	C
1		Concentration (ng/μL)	Ratio 260/280
2	EB	0.0	-0.18
3	T7-mut-sfGFP #4	690.2	1.90
4	EB #2	-0.3	2.92

5μL Cell free reactions of pY71-sfGFP and T7-mut-sfGFP were set up (25μL aliquoted into 5μL reactions). The plate reader was set up to read fluorescence 480/510 at sensitivity 19 and 30O.C. The plate was a 96 well V-bottomed plate with a membrane.

(2X dilution of Maxiprep 5 μL water and 5μL DNA)

No DNA (N)

Wells B2-B5
10μL Premix
9μL Extract

6μL Water

Blank (Water)

Wells C6-C9

pY71-sfGFP (P)

189ng/μL

Wells B6-B9

10μL Premix

9μL Extract

3μL DNA

3μL Water

T7-mut-sfGFP (T)

Wells C2-C5

10μL Premix

9μL Extract

1.6μL DNA

4.4μL Water

25μL lyophilization reactions were done. They were same cell free reactions as the ones run through the plate reader with 3reactions of No DNA, 3 reactions of pY71-sfGFP (P), and 3 reactions T7-mut-sfGFP (T).25μL cell free reactions aliquoted into 2.5μL reactions were also run on the paper-based sensor. There were two 25μL reactions of No DNA, one of pY71-sfGFP and one of T7-mut-sfGFP. There were 2 coupons each divided into 15 areas with 500μL of water. The coupon with the green sticker was for pY71-sfGFP, and the other with a purple sticker was for T7-mut- sfGFP. A 2.5μL reaction was pipetted into each of the 15 areas as shown below.

Made overnights of BL21-DE3 glycerol stocks (B1-B4) from 7/12/19. Miniprepped pY71-bFMO (NEB5) overnights, but only the first triplicate of each.

Nanodrop Results of pY71-bFMO (1st Triplicate Only)

	A	B	C	D
1		Concentration (ng/μL)	Ratio 260/280	Sequence #
2	EB #1	0.0	0.16
3	pY71-bFMO Glycerol Stock #1	38.0	1.97	BGC 277
4	pY71-bFMO Sample 1Triplicate 1	119.5	1.87	**BGC 278
5	pY71-bFMO Sample 2Triplicate 1	28.2	1.89	BGC 279
6	pY71-bFMO Sample 3Triplicate 1	94.0	1.73	BGC 280
7	EB #2	0.2	-1.25

**BGC 278 sequence was confirmed as the correct bFMO sequence.

Wednesday

T7-mutated-pY71-sfGFP overnights grew. 100μL of each overnight were measured in the plate reader (blanks of LB only were included). The settings included fluorescence 480/510, and sensitivity at 6 which is the default for the plate reader.
The overnights of pY71-bFMO BL21-DE3 were mini prepped to check for mutation (all glycerol stock B1-B4 had the mutation). B1-B3 had a pink pellet and B4 had a blue pellet.

Nanodrop Results of pY71-bFMO BL21-DE3 (B1-B4)

	A	B	C	D
1		Concentration (ng/μL)	ratio 260/280	Segment # (F primer only)
2	EB #1	-0.8	2.04
3	B1	168.3	1.91	BGC 281
4	B2	177.6	1.91	BGC 282
5	B3	164.0	1.91	BGC 283
6	B4	189.0	1.91	BGC 284
7	EB #2	-0.1	-31.27

The indigo standard curve was remade with 4.2mg into 420μL of DMSO. The 1st tube had 60μL of indigo. The 2nd, 3rd, 4th, etc. tube had 30μL of DMSO. 30μL of indigo from tube 1 was put into tube 2. 30μL of tube 2 was put in tube 3, etc until liquid became clear. Each was the put into the plate from B2-B17, C2-C17, and D2-D17 (C and D are triplicates) with read absorbance on the plate reader at 620nm.LabPats made a fluorescein 25μL standard curve. First a 10x dilution of 100μM stock of fluorescein needed to be done. 20μL stock into 180μL of 1X PBS were put into tube 1. The same dilution procedure as the indigo standard curve was used for this dilution. Each of the following tubes (2-6) begins with 100μL of 1X PBS and tube 7 has 100μL of 1X PBS only. 25μL of each tube was put into a well (tube one corresponds with well B2) in a clear v-bottom 96 well plate. The setup was B2-B8, C2-C8 and D2-D8 (C and D are triplicates). 2 reads of the plate were done one at sensitivity 19 and the other at sensitivity 6 (default). Both were read at 480/510.The pY71-sfGFP and T7-mut-pY71-sfGFP were lyophilized with 25μL water. They were then read in a 384 well plate. Wells B2- B4 had T7-mut-pY71-sfGFP, wells C2-C4 had pY71-sfGFP, and wells D2-D4 had no DNA. The fluorescence reading was 480-510. However the purple adapter (for absorbance) was left in so they must be redone.The overnights from 8/13 were read for absorbance as well. They were put in a 96-well plate with a flat, clear bottom. Needed a 2X dilution of the overnights (50μL of overnight and 50μL of LB). Two plates were run: one at an absorbance of 600nm and the other at an absorbance of 620nm. Only the pY71-sfGFP ones were read so this too needs to be redone. Glycerol stocks were made for the pY71-sfGFP overnights. They are being stored in the iGEM 2019 box labeled not recommended for use. LabPats also made a 200mL overnight of pY71-bFMO GnSn NEBS Sam1 Trp1 to maxiprep. It consisted of 200mL LB and 200μL of 50 Kan.

Thursday

LabPats maxi prepped pY71-bFMO G.S. NEB5 Sample 1 Triplicate 1. It is currently being stored in lambda box.

Nanodrop Results of pY71-bFMO Maxiprep

	A	B	C
1		concentration (ng/μL)	ratio 260/280
2	EB #1	0.0	0.17
3	pY71-bFMO Sam1 Trp1	148.3	1.9
4	EB #2	-0.2	-0.55

A mutagenesis was performed on the pY71-bFMO G.S. NEB5 Sample 1 Triplicate. 1, however it needs to be redone because the wrong primers were used.The 25μL fluorescence standard curve, and the lyophilization of T7-mut-pY71-sfGFP and pY71-sfGFP from the 14th were redone. The standard curve was done in a 384 well plate. Overnights consisting of 3mL of LB, 3μL of 50 Kan, and pY71-sfGFP glycerol stock #4. and overnights with T7-mut-pY71-sfGFP glycerol stock #4 were made.

Friday

Measured overnights of pY71-sfGFP + T7mut-pY71-sfGFP. A 2X dilution was done to fit the curve with 50 LB and 50 of the overnight. Both fluorescence and absorbance was measured with the plate reader settings at 480/510 for fluorescence and abs. at 600 nm. The wells used were B2-B4 for pY71-sfGFP, B5-B7 for T7mut-pY71-sfGFP, and B8-B10 for Blanks with just LB.Rehydrated the lyophilization of pY71-sfGFP and T7mut-sfGFP with 25 μL of water. A 384 well plate was used. Bubbling occurred in some of the wells. B2-B4 had No DNA, B5-B7 had pY71-sfGFP, and B8-B10 had T7mut-pY71-sfGFP. Triplicate 2 was placed in C8 well instead of B8 so an endpoint read was done on that well.LabPats also performed mutagenesis of pY71-bFMO to correct the T7 promoter.

Master Mix
5 iProof Buffer- 30μL

DNA
T7mut-F- 3μL

T7mut-R- 3μL
DNT p- 3μL
H2O- 105 μL
iProof polymerase- 3μL

10x dilution of primers- 1 μL of each primer- 9 μL of water

pY71-bFMO Sample 1: Triplicate 1- 119.5 ng/μL is about 120 ng/μL

5 ng/μL= 1:24 (23 μL) of water
10 ng/μL= 1:12 (11 μL) of water
20 ng/μL= 1:6 (5 μL) of water

50 ng/μL= 1: 2.4 (1.4 μL) of water

Transformed and left on plates on bench over the weekend. Also transformed pY71-bFMo into BL21 cells, but needs to beredone because it was meant to be BL21-DE3 cells.

8/19/2019 - 8/23/2019

Monday

Blanks and pY71-bFMO BL21 plates grew, but the rest did not. They were put into the 37°C incubator for a couple of hours. pY71-bFMO (sample 1 triplicate 1) was transformed into BL21-DE3 cells, plated and put in incubator overnight.Minipreps #2 that was sequenced on 8/12/19 was nanodropped and used in the ChrP bFMO and ChrP-GFPa1 RBS mutagenesis.

Nanodrop Results of ChrP-GFPa1 and ChrP-bFMO

	A	B	C
1		Concentration (ng/μL)	260/280
2	EB #1	-0.8	1.22
3	ChrP-GFPa1	156.3	1.90
4	ChrP-bFMO	311.4	1.90
5	EB #2	-0.7	0.94

Mixes
2-10 ng DNA
1 μM primer A- 0.5 μL
1 μM primer B- 0.5 μL
200 μM DNTPs- 0.5 μL
3U enzyme (iProof polymerase)- 0.5μL Buffer (HF)- 10 μL
Water- To 50 μL total

DNA in 5ng, 10ng, 20ng GFPa1
5 ng- 1:31.2 (30.2 μL water)

10 ng- 1:15.6 (14.6 μL water)

20 ng- 1: 2.8 (6.8 μL water)

Master Mix (n=4)

GFPa1

RBS_F_GFPa1... 2 μL

RBS_R... 2 μL

DNTP... 2 μL

Polymerase... 2 μL

Buffer... 40 μL

Water... 148 μL (196 μL/4)= 49 μL

bFMO
5 ng- 1:62.2 (61.2 μL water)

10 ng- 1:31.3 (30.1 μL water)

20 ng- 15.55 (14.55 μL water)

bFMO

RBS_F_bFMO... 2 μL

RBS_R... 2 μL

DNTP... 2 μL

Polymerase... 2 μL

Buffer... 40 μL

Water... 148 μL (196 μL/4)= 49 μL

Added 1 μL of DNA to each reaction except blank PCR cycle GFPa1

1 through 3- 12x

1. 95°C 5 min
2. 95°C 5 min
3. 60°C 1 min
4. 72°C 75 sec
5. 46°C 1 min
6. 72°C 7.5 min

1 through 3- 12x

1. 95°C 5 min
2. 95°C 1 min
3. 58°C 1 min
4. 72°C 100 sec
5. 46°C 1 min
6. 72°C 7.5 min

Added 0.5 μL of DpNI, incubated at 37°C for 2 hours,
Made overnights of 3OC12 circuit (PLas_sfGFP from JD #5) to take pictures of. Three overnights had 3OC12 and consisted of 3μL of 3OC12 + 3 μL chloramphenicol. Three other overnights had 3μL of DMSO + 3 μL of chloramphenicol.
Colonies of T7mut-bFMO grew in plates. LabPats used 20 ng, 5 μL plate. 2 colonies were made into 200 mL overnights to maxiprep, and 4 colonies were made into 3 μL overnights to miniprep. Plates that weren't used were left in the incubator overnight.

Tuesday

pLas- sfGFP overnights grew and T7mut-bFMO maxiprep and miniprep overnights grew. LabPats began transforming ChrP- GFPa1 and ChrP-bFMO RBS MUT Genes
1.) 100 μL of DH5∝
2 μL DNA20 uL 5x KCM
78 uL H2O (Nuclease free)
2.) Ice 30 minutes
3.) Heat shock 45 seconds @ 42°C
4.) Ice 2 minutes
5.) + 900 uL LB
6.) Incubate for 30 minutes
7.) Spin at 800 RPM for 5 minutes
8.) Took 900 uL out
9.) Resuspend in 200 uL
10.) Plate 100 uL and put in incubator @ 37°CMaxiprepped T7-mut-pY71-bFMO colony 5 and 6. Forgot to make immediate glycerol stocks so three overnights were made using the spun down pellet. Two overnights had 3mL LB, 3uL Kan, and the pellet. There was one blank with LB.

and stored PCR in 4°C fridge.

Nanodrop Results of Miniprepped T7-mut-bFMO Colonies 5 and 6

	A	B	C
1		Concentration (ng/uL)	260/280
2	EB #1	-0.3	-8.18
3	T7 mut-pY71- bFMO col. 5	576.2	1.89
4	T7 mut-pY71- bFMO col. 6	877.5	1.89
5	EB #2	-0.6	1.01

Miniprepped T7-mut-bFMO Colony 1-4 Overnights. Followed Protocol from 7/17/19. They are labeled MP1 T7mut-bFMO 8/20/19... MP4 T7mut-bFMO 8/20/19 and are in Lambda box.

Nanodrop Results of Miniprepped T7-mut-bFMO Colonies 1-4

	A	B	C
1		Concentration (ng/uL)	260/280
2	EB #1	1.1	0.93
3	T7mut- pY71- bFMO Col 1	157.9	1.87
4	T7mut- pY71- bFMO Col 2	150.8	1.88
5	T7mut- pY71- bFMO Col 3	150.2	1.88
6	T7mut- pY71- bFMO Col 4	102.7	1.89
7	EB #2	0.4	0.48

Sequencing Results

Miniprep 1- BGC285
Miniprep 2- BGC286

Miniprep 3- BGC287

Miniprep 4- BKC605

Miniprep 5- BGC606 Sequencing Confirmed

Miniprep 6- BGC607 Sequencing Confirmed

10x dilution of maxipreps: 1uL of maxiprep, 9uL H2O, F Primer only (because it’s a 10x dilution), 2.5uL P and 22.5 uL H2O.

Wednesday

25uL cell free reactions (T7mut-bFMO Colony 5) Indole- 0.0085 g to 1.7 mL DMSO is about 5mg/uL

A. No DNA (run in triplicates)

10 uL Premix
9 uL Extract
6 uL Water

B. DNA+Trp

10 uL Premix

9 uL Extract

2.5uL Trp

1uL DNA
2.5 uL Water

C. DNA + Indole

10 uL Premix
9 uL Extract

2.5uL indole

1uL DNA
2.5 uL Water

D. DNA + DMSO

10uL Premix

9uL Extract
1uL DNA

0.5uL DMSO

4.5uL Water

E. Trp
10uL Premix

9uL Extract

2.5uL Trp
3.5uL Water

F. Indole

10uL Premix

9uL Extract

2.5uL Indole

3.5uL Water

Used Wells I2-I23. I1 and I24 were blank (had nothing in them).

Transformed T7mut-bFMO maxiprep colony 5 into BL21-DE3 and set up overnights of ChrP-bFMO and ChrP-GFPa1.

Sequencing

ChrP-GFPa1 #1BKC608

ChrP-bFMO #1BKC609

Thursday

LabPats worked on measurement write up. The sequencing results came back, and it seems that colonies were switched. 4 colonies of GFPa1 grew and 1 colony of bFMO. The RBS was successfully put into ChrP-GFPa1 which was originally labeled as ChrP-bFMO #1. The RBS was missing the first “A” in ChrP-bFMO which was originally labeled ChrP-GFPa1 #1

Transformed ChrP-GFPa1 (sequencing confirmed) and ChrP-RBS-bFMO into BL21-DE3 cells

Cell free reactions from 8/21/19 of T7mut-bFMO at different conditions did not work. DNA + indole and indole reactions were high. It was realized that there was no DNA only condition so a DNA titration was set up.

DNA Titrations

No DNA
10μL Premix

9μL Extract

6μL Water

5.13μL Water

DNA (20 ng/μl)

10μL Premix

9μL Extract

0.87μL DNA

4.3μL Water

DNA (40 ng/μl)

10μL Premix

9μL Extract

1.7μL DNA

DNA (60 ng/μl)

10μL Premix

9μL Extract

2.6μL DNA

3.4μL Water

DNA (80 ng/μl)

10μL Premix

9μL Extract

2.6μL DNA

2.5μL Water

Set up overnights of T7-ChrP-GFPa1 and T7-ChrP-bFMO from plates

A: T7-ChrP-GFPa- 17-22-19- col.1BL21 (DE3)

B: T7-ChrP-bFMO- 7-22-19- col.1BL21 (DE3)

C: T7-ChrP-RBS GFPa1- 8-20-19- col.1DH5∝

D: T7-RBS-bFMO- 8-20-19 DH5∝

E: T7-ChrP-GFPa1- 8-9-19- col.1NEB5∝

F: T7-ChrP-bFMO- 8-9-19- col.1NEB5∝

G: T7-mut-bFMO- 8-21-19- BL21 (DE3)

Table of What Makes Up Each Reaction

	A	B	C	D	E	F	G
1			Media	Kan	Trp	IPTG	Colony (dissolved in 25 ul LB)
2	A	3x	3 mL	3			2
3	B	3x	3 mL	3			2
4		3x IPTG	3 mL	3		1.5	2
5		3x Trp	2.4 mL	3	600		2
6		3x IPTG +TRP	2.4 mL	3	600	1.5	2
7	C	3x	3 mL	3			2
8	D	3x	3 mL	3			2
9		3x Trp	2.4 mL	3	600		2
10	E	3x	3 mL	3			2
11	F	3x	3 mL	3			2
12		3x Trp	2.4 mL	3	600		2
13	G	3x	3 mL	3			2
14		3x IPTG	3 mL	3		1.5	2
15		3x Trp	2.4 mL	3	600		2
16		3x IPTG + Trp	2.4 mL	3	600	1.5	2

Friday

LabPats continued data analysis including on graphpad. Looking at results, only T7mut with and without the Trp worked (turned blue). No GFPa1 circuit showed fluorescence. The transformation of ChrP-bFMO and ChrP-GFPa1 worked. The ChrP- bFMO was grown on a 20ng Kan plate.bFMO extraction
50 μL of overnights into 750 μL of DMSO (μg/(μL DMSO))*(750μ L DMSO/50 μL culture) Multiply raw data by 15 (because 750/50)
Convert using the standard curve for indigo where 5.30E-5 conversion factor for indigo.

Plate Setup for bFMO absorbance measurement. (384 well plate with 25μl wells)

B2-B4- T7mut bFMO + Trp
B5-B7- T7 mut bFMO
B8-B10- Chrp bFMO +Trp

B11-B13 Chrp bFMO

B14-B16 DMSO only