Team:Alabama/Notebook
On May 22, 3 tubes of 5 mL of LB were inoculated with JM109, BL21(DE3) and NEB5α at 37 °C and 200 RPM. On May 28, the same strains were inoculated, but BL21(DE3) may not have worked. On May 29, 40 mM and 4 mM working stocks of gemcitabine were made (263.19 g/mol). 9 more cultures were inoculated (each strain with 10,000 x 40 mM in 5 mL, 1000 x 4 mM in 5 mL, and no gem in 5 mL). The following morning, the OD (10x dilution) of each sample was taken.
Strain
Drug
OD
JM109
No Gemcytabine
3.64 ng/mL
JM109
4mM
3.49 ng/mL
JM109
40mM
3.51 ng/mL
BL21(DE3)
No Gemcytabine
3.41 ng/mL
BL21(DE3)
4mM
3.71 ng/mL
BL21(DE3)
40mM
3.35 ng/mL
NEB5α
No Gemcytabine
3.32 ng/mL
NEB5α
4mM
3.14 ng/mL
NEB5α
40mM
3.48 ng/mL
On June 4, 5 mL of LB was inoculated with JM109, BL21(DE3), and NEB5α. Working stocks of 4 mM and 40 mM PTX were made. That evening, 9 more culture tubes were inoculated the same as before but with PTX. They all have 50 μL of target strain. 40 mM appeared to have precipitated.
On the morning of June 5, all 9 tubes grew and the 40 mM precipitate was gone. The OD600 was taken with a 10x dilution.
Strain
Drug
OD
JM109
No PTX
2.45 ng/mL
JM109
4mM
2.71 ng/mL
JM109
40mM
2.50 ng/mL
BL21(DE3)
No PTX
2.46 ng/mL
BL21(DE3)
4mM
2.35 ng/mL
BL21(DE3)
40mM
2.29 ng/mL
NEB5α
No PTX
2.9 ng/mL
NEB5α
4mM
2.83 ng/mL
NEB5α
40mM
2.56 ng/mL
HPLC samples were prepared with 4 mM drug and LB on June 12. Potential problems could be not enough time, wrong wavelength, or too low of a concentration.
LB
LB+PTX
LB+Gem
Gemcytabine test
Noisy fail
Noisy fail
Noisy fail
15% MeOH 20 min
Normal
Normal LB, no extra peaks
Normal LB, no extra peaks
15% MeOH 30 min
normal
Normal LB, no extra peaks
Normal LB, no extra peaks
15% MeOH 30 min
225-300 nm
Normal
Normal LB, no extra peaks
Normal LB, no extra peaks
New HPLC samples were prepared at 20 μM instead of 4 μM with the same protocol. The samples were run the next day with no change in peaks. On June 17, LB+20 μM solution at 30% B was run for 20 minutes with inconclusive results. A flow rate of 1 mL/min was tried but it leaked. On June 18, the same sample with a flow rate of 0.7 mL/min was run and the results were inconclusive. This was the highest possible flow rate without leaking.
3 mL of LB were inoculated with 3 strains of E. coli (JM109, BL21(DE3), and NEB5α on June 19. The 40% B HPLC sample was ran for 20 min at 0.7 mL/min. BL21(DE3) was inoculated into the following concentrations: no drug, 20 μM, 200 μM Gem, 1000 μM Gem, 20 μM PTX, 200 μM PTX, and 1000 μM PTX at 37 °C and 200 RPM. All of the PTX samples precipitated immediately. The OD Calibration from iGEM was performed. All 7 samples of BL21(DE3) grew well. The 200 μM and 1000 μM PTX samples still had chunky precipitate. The samples were stored in the 4 °C fridge for future testing. The new HPLC samples of LB+Gem at 0 μM, 20 μM, 200 μM, and 1000 μM were prepared at 10% MeOH, 1 mL/min per Singh et al, 2015. There were peaks at 2.25 min.
On June 21, HPLC samples of LB, 200 μM Gem, 1000 μM Gem, LB+DMSO, and pure MeOH were ran at 7.5%, 0.5 mL/min for 15 minutes. On June 22, LB, 200 μM Gem, 1000 μM, LB+DMSO, cell culture (CC) LB+200 μM Gem, CC LB+1000 Gem, CCLB were ran at 5%, 0.5 mL/min for 30 minutes.
On June 26, samples were prepared for a calibration curve. New working stocks of 4 mM Gem was made from DMSO and 40 mM stock. Concentrations of 0, 50 μM, 100 μM, 200 μM, 500 μM, and 1000 μM were made with LB and MeOH.
BC21(DE3) was inoculated to 3 mL LB on July 1. The following morning 5 mL LB was inoculated with 50 μL seed in the morning and a growth curve with samples taken in triplicate every 3 hours was performed.
Time
Blank
1
2
3
7:30 am
0
0.014
0.015
0.015
10:30 am
0
0.285
0.290
0.373
1:30 pm (5x)
0
0.495
0.480
0.470
4:40 pm (5x)
0
0.66
0.710
0.700
7:30 pm (5x)
0
0.905
0.965
0.930
10:30 pm (5x)
0
1.02
0.995
1.00
1:30 am (5x)
0
1.01
1.035
0.980
7:30 am (5x)
0
1.065
1.02
1.075
Then, HPLC samples were made from the growth curve. New MeOH 100 and 200 μM samples were made to compare to calibration curve. On July 8, MeOH calibration samples were made with 50, 100, 200, 500, and 1000 μM PTX. BL21(DE3) from glycerol stock was inoculated into LB.
The next day the samples were washed with M9 minimal and spun for 2 minutes each time. 200 μM Gem, 200 μM PTX precipitated, and no drug were inoculated into M9 minimal. On July 10, there was good cell growth for M9 no drug, but no cell growth for PTX or 200 μM Gem. Then, the samples were inoculated with just DMSO. On July 11, there was no growth in 200 μM Gem, some growth in M9+DMSO, and some growth and precipitate in 200 μM PTX. By July 12, the gemcitabine culture finally started to grow, and on July 14, there was significant cell growth in 7/9/19 200 μM Gem sample.
On the night of July 17, 3 mL LB inoculated with BL21(DE3) at 37 °C and 200 RPM. This inoculum was used to inoculate 5 μL into 3 mL fresh LB. That evening, 5 μL of previous inoculum was inoculated in 25 mL fresh LB. The sample was washed with M9 minimal three times, then spun for 25 minutes at 13000 xg at 4 °C. Then 5 μL of washed cells were inoculated into 25 mL M9 media and grown overnight. The OD600 concentrations the following morning were 6.65 ng/mL for LB and 1.71 ng/mL for M9. 250 μL of overnight LB were used to inoculate 25 mL fresh media, then were resuspended in 1 mL KPi. The OD values were 34.4 ng/mL for MP and 73.2 ng/mL for LB.
On July 22, a plate of Nissle 1917 was received from UAH. 4 colonies were seeded. Glycerol stocks were made with 4 colony tubes. The starting ODs (10x dilution) were 3.28, 3.73, 3.42, and 3.20. The cells from tube 4 appeared the least smudged on plate, so they will be used for testing. 2 mL LB was inoculated with Nissle 1917(4), then washed three times with M9. 5 μL was inoculated into 25 mL new M9 minimal. On July 24, the OD (10x dilution) was 2.55. 3 mL LB was inoculated with Nissle 1917(4) and a growth curve was performed on July 25.
M9
Gemcytabine
0 Minutes
0.005, 0.004, 0.007
0.002, 0.003, 0.001
60 Minutes
0.014, 0.014, 0.016
0.011, 0.013, 0.012
120 Minutes
0.026, 0.025, 0.026
0.019, 0.022, 0.022
180 Minutes
0.048, 0.048, 0.05
0.03, 0.034, 0.032
240 Minutes
0.091, 0.093, 0.095
0.049, 0.052, 0.051
300 Minutes
0.156, 0.162, 0.163
0.059, 0.065, 0.066
360 Minutes
0.231, 0.242, 0.245
0.068, 0.072, 0.072
540 Minutes
0.883, 0.685, 0.685
0.194, 0.1168, 0.161
720 Minutes
0.575, 0.570, 0.58
0.33, 0.325, 0.32
900 Minutes
0.64, 0.57, 0.6
0.535, 0.545, 0.505
1080 Minutes
0.64, 0.555, 0.605
0.585, 0.56, 0.57
1260 Minutes
0.61, 0.625, 0.615
0.635, 0.66, 0.475
1530 Minutes
0.545, 0.515, 0.565
0.595, 0.635, 0.625
3 mL LB inoculated with Nissle 1917(4) from glycerol stocks. 4 hours later, 5 μL was inoculated in 25 mL M9.
On July 26, a resting cell assay was performed with Nissle 1917(4). The starting OD was 2.97, and the OD with KPi was 65.9.
On August 6, 1 mL M9 minimal was inoculated with Nissle 1917(4). 8 hours later there was no growth. 25 hours after the inoculation there was growth in M9. On August 11, 3 mL LB was inoculated with JM109. 200 μL JM109 was inoculated with 20 mL LB the next day. A chemically competent ally was made.
Biobrick parts with 10 μL water
K1692032
K1033900
K1033903
K1033907
K1033914
K1033923
K1033912
K1033926
K1033928
K1073022
K1073024
K1073026
K1033282
The bacteria were chemically transformed to heat shock into chemically competent JM109. They were then plated 90/10 and incubated at 37 °C. A resting cell assay was performed, but the data was bad. On August 13th, the plates were checked. 19-6-15B, 19-5-10B, 19-6-9F, and 19-6-9H did not grow. 19-6-13D had no color. 19-6-5P might not have been colored correctly. 5N was blue, 7B was supposed to be yellow but wasn’t, 7F was green, 7L was purple, 9D and 11P were purple, and 13B was the regular color. 5P was very light purple but did not grow on 10% plate, 9F did not grow well on the first check and had no color. 10B did not grow well on the first check and had no color. There were some giant colonies and lots of tiny colonies. 13D had no color. 1 colony per sample was inoculated in 3 mL LB+Chl34 for a glycerol stock.
A PCR was performed using P1-F1/P1-R1 and PUC19, as well as P3-F1/P3-R1 and pet28.
All grew except K1692032 (5-10B) and K10339261 6-9F. Start glycerol stocks then check back on last 2. 1 hour later 5-10B and 6-9F not grown yet, allowing to grow overnight.
8/14/19
Same PCR as yesterday with temperature gradient. Not successful, used new samples of gradient and ran samples from 8/13 by loading all 12 samples in SyberSafe stained gel.
Inoculated 2mL of M9 with Nissle 1917(4) from glycerol stock. Used 2 mL of m9 and 5 microliters of seed nissle from 8/12. Reinoculated 3mL fresh LB+Chl134 with 30 microliters of K1033926 and K1692032. Both appear to be visibly pink/blue.
Redid heat shock for K1033928 (9H) and K1033282 (15B), then plated at 10%/90% in incubator at 37 degrees celsius.
Redid gradient PCR and ran.
8/15/19
Inoculate 2.5 microliter seed culture to 25 mL m9 minimal media. Growth Curve data confirmed growth of C1, C2, C3, G1, G2, and G3.
8/17/19
Prepared gibson assembly as follows for 1100, 2200, and 3300. Samples then heat shocked to combine with Ecloni and plated them and allowed to grow overnight.
8/18/19
No growth on any plates from gibson assembly.
8/20/19
Made 100 and 200 micro molar Gemcitabine stock. Then placed in shaker and took HPLC samples at both t=0 and t=2 hours
9/10/19
Ran PCR with new primers as specified.
5X HF Buffer
5 μL
10 μM DNTPs
0.5 μL
10 μM F Primer
1.25 μL
10 μM R Primer
1.25 μL
Phusion
0.25 μL
Template DNA
0.5 μL
Water
16.25 μL
Total
25 μL
9/17/19
Ran gel on PCR done 9/10/19, bands were present.
9/18/19
Ran PCR gradient using same parameters from 9/10/19 on same 7 samples (1100 A, 1100 B, 2200 A, 2200 B, 2200 C, 3300B, 3300 C). However, 25 μL of each sample was aliquoted into 5 flasks in order to determine which temperature had maximum effectiveness.
9/20/19
Ran gel on PCR from 9/18/19 and no bands were shown.
9/23/19
Ran PCR using same parameters form 9/18/19.
9/25/19
Ran gel on PCR from 9/23/19, all bands shown. Scaled up to 50 μL with all samples except for 3300 B as it had some indescrepencies in size with what was expected. Temperature used for the scale up was 69.4 degrees celsius.
9/26/19
Performed cycle pure on 6 samples from PCR on 9/25/19, DNA concentrations below.
H20
2.052
1.900
1100 A
10.108
19.228
1100 B
50.008
24.472
2200 A
3.800
12.464
2200 B
15.808
56.240
2200 C
20.900
30.932
3300 C
29.944
25.080
Ran PCR on the 6 samples with varying amounts to make a 50 nanogram concentration.
Ran gel on PCR products to determine effectiveness, discovered discrepancies in 2200 A, 2200 B, and 3300 C.
10/1/19
Reran gel on 3 questionable samples and received good results with 2200 A and 3300 C, however 2200 B needed to be remade. Therefore PCR was rerun with 2200 B, with 100 μL being split in for 25 μL tubes at 69.4 degrees celsius.
10/2/19
Combined samples from 10/1/19 in order to use them for a cyclopure.
10/3/19
Gibson assembly run on both 1100 and 2200 sample with amounts as follows.
H20
9.38 μL
1100 A
1.5 μL
PUC 19
1.45 μL
1100 B
3.67 μL
Enzyme
2 μL
Buffer
2 μL
Total
20 μL
H20
3.05 μL
2200 A
4 μL
2200 B
2.7 μL
2200 C
6.25 μL
Enzyme
2 μL
Buffer
2 μL
Total
20 μL
With both reactions pared down to 10 μL.
10/7/19
Heat shocked Ecloni and put in both the 1100 and 2200 plasmids. Rescued cells for an hour then plated 1100 on LB+CHI37 and 2200 on LB+Kan with 20/80% ratios at 37 degrees celsius. Left plates overnight.
10/8/19
Checked plates and found growth on 1100 sample but not on 2200 sample. Redid gibson assembly on 2200 samples with amounts as follows.
H20
4.4 μL
2200 A
4 μL
2200 B
1.35 μL
2200 C
6.25 μL
Enzyme
2 μL
Buffer
2 μL
Total
20 μL
10/14/19
Performed heat shock with Ecloni and put in 2200 sample. Rescued cells for an hour then plated on LB+Kan at a 20/80% at 37 degrees celsius and left plates overnight to grow.
On May 22, 3 tubes of 5 mL of LB were inoculated with JM109, BL21(DE3) and NEB5α at 37 °C and 200 RPM. On May 28, the same strains were inoculated, but BL21(DE3) may not have worked. On May 29, 40 mM and 4 mM working stocks of gemcitabine were made (263.19 g/mol). 9 more cultures were inoculated (each strain with 10,000 x 40 mM in 5 mL, 1000 x 4 mM in 5 mL, and no gem in 5 mL). The following morning, the OD (10x dilution) of each sample was taken.
| Strain | Drug | OD |
|---|---|---|
| JM109 | No Gemcytabine | 3.64 ng/mL |
| JM109 | 4mM | 3.49 ng/mL |
| JM109 | 40mM | 3.51 ng/mL |
| BL21(DE3) | No Gemcytabine | 3.41 ng/mL |
| BL21(DE3) | 4mM | 3.71 ng/mL |
| BL21(DE3) | 40mM | 3.35 ng/mL |
| NEB5α | No Gemcytabine | 3.32 ng/mL |
| NEB5α | 4mM | 3.14 ng/mL |
| NEB5α | 40mM | 3.48 ng/mL |
On June 4, 5 mL of LB was inoculated with JM109, BL21(DE3), and NEB5α. Working stocks of 4 mM and 40 mM PTX were made. That evening, 9 more culture tubes were inoculated the same as before but with PTX. They all have 50 μL of target strain. 40 mM appeared to have precipitated.
On the morning of June 5, all 9 tubes grew and the 40 mM precipitate was gone. The OD600 was taken with a 10x dilution.
| Strain | Drug | OD |
|---|---|---|
| JM109 | No PTX | 2.45 ng/mL |
| JM109 | 4mM | 2.71 ng/mL |
| JM109 | 40mM | 2.50 ng/mL |
| BL21(DE3) | No PTX | 2.46 ng/mL |
| BL21(DE3) | 4mM | 2.35 ng/mL |
| BL21(DE3) | 40mM | 2.29 ng/mL |
| NEB5α | No PTX | 2.9 ng/mL |
| NEB5α | 4mM | 2.83 ng/mL |
| NEB5α | 40mM | 2.56 ng/mL |
HPLC samples were prepared with 4 mM drug and LB on June 12. Potential problems could be not enough time, wrong wavelength, or too low of a concentration.
| LB | LB+PTX | LB+Gem | |
|---|---|---|---|
| Gemcytabine test | Noisy fail | Noisy fail | Noisy fail |
| 15% MeOH 20 min | Normal | Normal LB, no extra peaks | Normal LB, no extra peaks |
| 15% MeOH 30 min | normal | Normal LB, no extra peaks | Normal LB, no extra peaks |
| 15% MeOH 30 min 225-300 nm |
Normal | Normal LB, no extra peaks | Normal LB, no extra peaks |
New HPLC samples were prepared at 20 μM instead of 4 μM with the same protocol. The samples were run the next day with no change in peaks. On June 17, LB+20 μM solution at 30% B was run for 20 minutes with inconclusive results. A flow rate of 1 mL/min was tried but it leaked. On June 18, the same sample with a flow rate of 0.7 mL/min was run and the results were inconclusive. This was the highest possible flow rate without leaking.
3 mL of LB were inoculated with 3 strains of E. coli (JM109, BL21(DE3), and NEB5α on June 19. The 40% B HPLC sample was ran for 20 min at 0.7 mL/min. BL21(DE3) was inoculated into the following concentrations: no drug, 20 μM, 200 μM Gem, 1000 μM Gem, 20 μM PTX, 200 μM PTX, and 1000 μM PTX at 37 °C and 200 RPM. All of the PTX samples precipitated immediately. The OD Calibration from iGEM was performed. All 7 samples of BL21(DE3) grew well. The 200 μM and 1000 μM PTX samples still had chunky precipitate. The samples were stored in the 4 °C fridge for future testing. The new HPLC samples of LB+Gem at 0 μM, 20 μM, 200 μM, and 1000 μM were prepared at 10% MeOH, 1 mL/min per Singh et al, 2015. There were peaks at 2.25 min.
On June 21, HPLC samples of LB, 200 μM Gem, 1000 μM Gem, LB+DMSO, and pure MeOH were ran at 7.5%, 0.5 mL/min for 15 minutes. On June 22, LB, 200 μM Gem, 1000 μM, LB+DMSO, cell culture (CC) LB+200 μM Gem, CC LB+1000 Gem, CCLB were ran at 5%, 0.5 mL/min for 30 minutes.
On June 26, samples were prepared for a calibration curve. New working stocks of 4 mM Gem was made from DMSO and 40 mM stock. Concentrations of 0, 50 μM, 100 μM, 200 μM, 500 μM, and 1000 μM were made with LB and MeOH.
BC21(DE3) was inoculated to 3 mL LB on July 1. The following morning 5 mL LB was inoculated with 50 μL seed in the morning and a growth curve with samples taken in triplicate every 3 hours was performed.
| Time | Blank | 1 | 2 | 3 |
|---|---|---|---|---|
| 7:30 am | 0 | 0.014 | 0.015 | 0.015 |
| 10:30 am | 0 | 0.285 | 0.290 | 0.373 |
| 1:30 pm (5x) | 0 | 0.495 | 0.480 | 0.470 |
| 4:40 pm (5x) | 0 | 0.66 | 0.710 | 0.700 |
| 7:30 pm (5x) | 0 | 0.905 | 0.965 | 0.930 |
| 10:30 pm (5x) | 0 | 1.02 | 0.995 | 1.00 |
| 1:30 am (5x) | 0 | 1.01 | 1.035 | 0.980 |
| 7:30 am (5x) | 0 | 1.065 | 1.02 | 1.075 |
Then, HPLC samples were made from the growth curve. New MeOH 100 and 200 μM samples were made to compare to calibration curve. On July 8, MeOH calibration samples were made with 50, 100, 200, 500, and 1000 μM PTX. BL21(DE3) from glycerol stock was inoculated into LB.
The next day the samples were washed with M9 minimal and spun for 2 minutes each time. 200 μM Gem, 200 μM PTX precipitated, and no drug were inoculated into M9 minimal. On July 10, there was good cell growth for M9 no drug, but no cell growth for PTX or 200 μM Gem. Then, the samples were inoculated with just DMSO. On July 11, there was no growth in 200 μM Gem, some growth in M9+DMSO, and some growth and precipitate in 200 μM PTX. By July 12, the gemcitabine culture finally started to grow, and on July 14, there was significant cell growth in 7/9/19 200 μM Gem sample.
On the night of July 17, 3 mL LB inoculated with BL21(DE3) at 37 °C and 200 RPM. This inoculum was used to inoculate 5 μL into 3 mL fresh LB. That evening, 5 μL of previous inoculum was inoculated in 25 mL fresh LB. The sample was washed with M9 minimal three times, then spun for 25 minutes at 13000 xg at 4 °C. Then 5 μL of washed cells were inoculated into 25 mL M9 media and grown overnight. The OD600 concentrations the following morning were 6.65 ng/mL for LB and 1.71 ng/mL for M9. 250 μL of overnight LB were used to inoculate 25 mL fresh media, then were resuspended in 1 mL KPi. The OD values were 34.4 ng/mL for MP and 73.2 ng/mL for LB.
On July 22, a plate of Nissle 1917 was received from UAH. 4 colonies were seeded. Glycerol stocks were made with 4 colony tubes. The starting ODs (10x dilution) were 3.28, 3.73, 3.42, and 3.20. The cells from tube 4 appeared the least smudged on plate, so they will be used for testing. 2 mL LB was inoculated with Nissle 1917(4), then washed three times with M9. 5 μL was inoculated into 25 mL new M9 minimal. On July 24, the OD (10x dilution) was 2.55. 3 mL LB was inoculated with Nissle 1917(4) and a growth curve was performed on July 25.
| M9 | Gemcytabine | |
|---|---|---|
| 0 Minutes | 0.005, 0.004, 0.007 | 0.002, 0.003, 0.001 |
| 60 Minutes | 0.014, 0.014, 0.016 | 0.011, 0.013, 0.012 |
| 120 Minutes | 0.026, 0.025, 0.026 | 0.019, 0.022, 0.022 |
| 180 Minutes | 0.048, 0.048, 0.05 | 0.03, 0.034, 0.032 |
| 240 Minutes | 0.091, 0.093, 0.095 | 0.049, 0.052, 0.051 |
| 300 Minutes | 0.156, 0.162, 0.163 | 0.059, 0.065, 0.066 |
| 360 Minutes | 0.231, 0.242, 0.245 | 0.068, 0.072, 0.072 |
| 540 Minutes | 0.883, 0.685, 0.685 | 0.194, 0.1168, 0.161 |
| 720 Minutes | 0.575, 0.570, 0.58 | 0.33, 0.325, 0.32 |
| 900 Minutes | 0.64, 0.57, 0.6 | 0.535, 0.545, 0.505 |
| 1080 Minutes | 0.64, 0.555, 0.605 | 0.585, 0.56, 0.57 |
| 1260 Minutes | 0.61, 0.625, 0.615 | 0.635, 0.66, 0.475 |
| 1530 Minutes | 0.545, 0.515, 0.565 | 0.595, 0.635, 0.625 |
3 mL LB inoculated with Nissle 1917(4) from glycerol stocks. 4 hours later, 5 μL was inoculated in 25 mL M9.
On July 26, a resting cell assay was performed with Nissle 1917(4). The starting OD was 2.97, and the OD with KPi was 65.9.
On August 6, 1 mL M9 minimal was inoculated with Nissle 1917(4). 8 hours later there was no growth. 25 hours after the inoculation there was growth in M9. On August 11, 3 mL LB was inoculated with JM109. 200 μL JM109 was inoculated with 20 mL LB the next day. A chemically competent ally was made.
Biobrick parts with 10 μL water
K1692032
K1033900
K1033903
K1033907
K1033914
K1033923
K1033912
K1033926
K1033928
K1073022
K1073024
K1073026
K1033282
The bacteria were chemically transformed to heat shock into chemically competent JM109. They were then plated 90/10 and incubated at 37 °C. A resting cell assay was performed, but the data was bad. On August 13th, the plates were checked. 19-6-15B, 19-5-10B, 19-6-9F, and 19-6-9H did not grow. 19-6-13D had no color. 19-6-5P might not have been colored correctly. 5N was blue, 7B was supposed to be yellow but wasn’t, 7F was green, 7L was purple, 9D and 11P were purple, and 13B was the regular color. 5P was very light purple but did not grow on 10% plate, 9F did not grow well on the first check and had no color. 10B did not grow well on the first check and had no color. There were some giant colonies and lots of tiny colonies. 13D had no color. 1 colony per sample was inoculated in 3 mL LB+Chl34 for a glycerol stock.
A PCR was performed using P1-F1/P1-R1 and PUC19, as well as P3-F1/P3-R1 and pet28.
All grew except K1692032 (5-10B) and K10339261 6-9F. Start glycerol stocks then check back on last 2. 1 hour later 5-10B and 6-9F not grown yet, allowing to grow overnight.
8/14/19
Same PCR as yesterday with temperature gradient. Not successful, used new samples of gradient and ran samples from 8/13 by loading all 12 samples in SyberSafe stained gel.
Inoculated 2mL of M9 with Nissle 1917(4) from glycerol stock. Used 2 mL of m9 and 5 microliters of seed nissle from 8/12. Reinoculated 3mL fresh LB+Chl134 with 30 microliters of K1033926 and K1692032. Both appear to be visibly pink/blue.
Redid heat shock for K1033928 (9H) and K1033282 (15B), then plated at 10%/90% in incubator at 37 degrees celsius.
Redid gradient PCR and ran.
8/15/19
Inoculate 2.5 microliter seed culture to 25 mL m9 minimal media. Growth Curve data confirmed growth of C1, C2, C3, G1, G2, and G3.
8/17/19
Prepared gibson assembly as follows for 1100, 2200, and 3300. Samples then heat shocked to combine with Ecloni and plated them and allowed to grow overnight.
8/18/19
No growth on any plates from gibson assembly.
8/20/19
Made 100 and 200 micro molar Gemcitabine stock. Then placed in shaker and took HPLC samples at both t=0 and t=2 hours
9/10/19
Ran PCR with new primers as specified.
| 5X HF Buffer | 5 μL |
|---|---|
| 10 μM DNTPs | 0.5 μL |
| 10 μM F Primer | 1.25 μL |
| 10 μM R Primer | 1.25 μL |
| Phusion | 0.25 μL |
| Template DNA | 0.5 μL |
| Water | 16.25 μL |
| Total | 25 μL |
9/17/19
Ran gel on PCR done 9/10/19, bands were present.
9/18/19
Ran PCR gradient using same parameters from 9/10/19 on same 7 samples (1100 A, 1100 B, 2200 A, 2200 B, 2200 C, 3300B, 3300 C). However, 25 μL of each sample was aliquoted into 5 flasks in order to determine which temperature had maximum effectiveness.
9/20/19
Ran gel on PCR from 9/18/19 and no bands were shown.
9/23/19
Ran PCR using same parameters form 9/18/19.
9/25/19
Ran gel on PCR from 9/23/19, all bands shown. Scaled up to 50 μL with all samples except for 3300 B as it had some indescrepencies in size with what was expected. Temperature used for the scale up was 69.4 degrees celsius.
9/26/19
Performed cycle pure on 6 samples from PCR on 9/25/19, DNA concentrations below.
| H20 | 2.052 | 1.900 |
|---|---|---|
| 1100 A | 10.108 | 19.228 |
| 1100 B | 50.008 | 24.472 |
| 2200 A | 3.800 | 12.464 |
| 2200 B | 15.808 | 56.240 |
| 2200 C | 20.900 | 30.932 |
| 3300 C | 29.944 | 25.080 |
Ran PCR on the 6 samples with varying amounts to make a 50 nanogram concentration.
Ran gel on PCR products to determine effectiveness, discovered discrepancies in 2200 A, 2200 B, and 3300 C.
10/1/19
Reran gel on 3 questionable samples and received good results with 2200 A and 3300 C, however 2200 B needed to be remade. Therefore PCR was rerun with 2200 B, with 100 μL being split in for 25 μL tubes at 69.4 degrees celsius.
10/2/19
Combined samples from 10/1/19 in order to use them for a cyclopure.
10/3/19
Gibson assembly run on both 1100 and 2200 sample with amounts as follows.
| H20 | 9.38 μL |
|---|---|
| 1100 A | 1.5 μL |
| PUC 19 | 1.45 μL |
| 1100 B | 3.67 μL |
| Enzyme | 2 μL |
| Buffer | 2 μL |
| Total | 20 μL |
| H20 | 3.05 μL |
|---|---|
| 2200 A | 4 μL |
| 2200 B | 2.7 μL |
| 2200 C | 6.25 μL |
| Enzyme | 2 μL |
| Buffer | 2 μL |
| Total | 20 μL |
With both reactions pared down to 10 μL.
10/7/19
Heat shocked Ecloni and put in both the 1100 and 2200 plasmids. Rescued cells for an hour then plated 1100 on LB+CHI37 and 2200 on LB+Kan with 20/80% ratios at 37 degrees celsius. Left plates overnight.
10/8/19
Checked plates and found growth on 1100 sample but not on 2200 sample. Redid gibson assembly on 2200 samples with amounts as follows.
| H20 | 4.4 μL |
|---|---|
| 2200 A | 4 μL |
| 2200 B | 1.35 μL |
| 2200 C | 6.25 μL |
| Enzyme | 2 μL |
| Buffer | 2 μL |
| Total | 20 μL |
10/14/19
Performed heat shock with Ecloni and put in 2200 sample. Rescued cells for an hour then plated on LB+Kan at a 20/80% at 37 degrees celsius and left plates overnight to grow.