Team:US AFRL CarrollHS/Notebook
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 ReaderSettings 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).- pLas_sfGFP miniprep TD#1
- pLas_sfGFP miniprep TD#2
- pLas_sfGFP miniprep TD#3
- pLas_sfGFP #6 JD
- pLas_sfGFP #3 HJ
- pLas_sfGFP #5 JD
- pLas_sfGFP #4 HJ
| 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 |
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 ReaderSettings 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 |
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 Reaction2μL 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 WaterTuesday
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 Premix9μL 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 July7/8/2019 - 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 |
| 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 |
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 |
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.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 IPTGB = 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.| 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 |
| 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 |
| 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-bFMORXN 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: PurpleC2, 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
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 prMaxiprep
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 plateMade 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'sThe 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
MaxiprepsCell 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. NaikRan 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
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.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 |
| 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 |
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 |
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 |
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 |
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 |
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 Mix5 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 |
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
- 95°C 5 min
- 95°C 5 min
- 60°C 1 min
- 72°C 75 sec
- 46°C 1 min
- 72°C 7.5 min
1 through 3- 12x
- 95°C 5 min
- 95°C 1 min
- 58°C 1 min
- 72°C 100 sec
- 46°C 1 min
- 72°C 7.5 min
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 Genes1.) 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 |
| 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 |
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/uL9 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 DNA10μL 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 extraction50 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