Inoculate three strains for the next manipulation of the week (test the oscillator system, competent cell preparation and cloning) using the protocol of paper Potvin-Trottier 2016.
Week 1
29.06.2020
Put the three strain with the double plasmids (all DHL708 strains) to culture o/n for for testing oscillations next day
30.06.2020
Measure fluorescence of each time point (o/n, T0, T1...) in a 96-well plate to test the oscillations. Followed the protocol and used media in Potvin-Trottier 2016. Dilutions done every 50 minutes from 10am till 5pm and fluorescence for each time point measured in plate reader
Week 2
06.07.2020
Put 2 strains (A2=DHL708 pLPT119 + pLPT 41 and A3=DHL708 pLPT107 + pLPT41) for O/N incubation, with goal to achieve OD = 0.2 in the next morning
Made o/n culture from it
07.07.2020
We looked at the growth overtime of the strain A2 and A3, to assess how much time is needed to go from 0,05 to 0,2. This was needed after seeing that our former Imaging media (IM) was at 2% glucose instead of 0.2% like the paper. Which explained in part why the serial dilution made on Monday 06.07.2020 did not work
08.07.2020
Made two types of experiments: growth and dilution on tubes and on plates
09.07.2020
Started to analyse the data collected Wednesday 08.07
Week 3
14.07.2020
We prepared some transformed cells for the non fluorescent control --> (pj2060 + p41 in both Nissle and DHL708)
16.07.2020
Redid fluorescence experiment with:
DHL708 pLPT119 + pLPT 41
DHL708 pLPT107 + pLPT41
DHL708 pJ2060 + pLPT41 (Non-fluorescent)
Redid all new all fresh Imaging media Stocks + made pluronic F to add to this media
Week 4
21.07.2020
Redid fluorescence experiment w/ DHL708 and both media to confirm + because some of the measurements messed up a bit last time
22.07.2020
Prepared fluorescence experiment for the 23.07.2020
23.07.2020
Redid fluorescence experiment w/ DHL708 pLPT119/107 + pLPT41 and Nissle ΔClb pLPT119 + pLPT41 with only Imaging media this time
24.07.2020
Made the dilution experiment to assess the growth of our 2 strains and establish a incubation protocol
Week 5
27.07.2020
Plate experiment with DHL708 (plasmid 107, 234, 41 and 145 combination and 119+mKate tested)
29.07.2020
Planned and prepared the the cultures for tomorrow's plate experiment
30.07.2020
Plate experiment (test of various external factors for the experiment in DHL708)
Week 6
04.08.2020
Fluorescence experiment with Nissle
o/n cultures for experiments tomorrow
05.08.20
Growth curve experiment with Nissle and Nissle+plasmids
06.08.2020
Set up o/n colonies to redo growth experiment
07.08.2020
Redid growth experiment with more time point measurements and following strains:
Nissle pLTP234 + pLPT41
Nissle pLTP234 + pLPT145 (simple and double transformant)
NEB5a + 234 +145
DHL708 pJ2060 + pLPT41 ( NF)
Week 7
12.08.2020
Planification of the plate experiment of tomorrow
13.08.2020
Redid growth experiment with more time point measurements and following strains:
Nissle pLTP234 + pLPT145
Nissle pLTP119 + pLPT41
Nissle pLPT119 + pLPT41-pelB-Azurin
Week 11
07.09.2020
Did o/n experiment of fluorescence by doing a dilution experiment
08.09.2020
Growth experiment for plasmid stability
Azurin Purification
We started our azurin experiments about 3 weeks into the lab access. We used the following plasmids for all these experiments:
Plasmid number
Plasmid name
Description
Resistance
Link to map
Plasmid 9
pLIBT7_A_peIB-Azurin-3C-TwinStrep-His10
plasmid expressing azurin with pelB-5D and 3X FLAG tag
Transformation of plasmid 9 into BL21 expression strain, left to grow over the weekend at room temperature, plated onto LBA + Amp
Week 4
21.07.2020
Set up 2 cultures of 15-20mL in TB media (see recipe in Protocols). Grown at 37°C in a shaking incubator until OD600 0.1-0.2
Directly induced one culture with 5uM IPTG
Left to shake for a further 2.5hr at 37°C, then harvested the pellet and froze at -20°C
Cooled the second culture down to 20°C, left to shake for 30 minutes and induced overnight with 5uM IPTG, left to incubate at the same temperature.
22.07.2020
Harvested pellet from culture 2 and proceeded with the Protein purification small-scale protocol and ran on 15% SDS-Page at the end, stained and destained
26.07.2020
Large-scale purification of azurin from BL21 attempted over the weekend without conclusive results
29.07.2020
Induction of azurin expression from plasmid pJ2060-pelB-azurin in Nissle 1917. Grown until OD600 0.1-0.2 in TB and induced with 0.2% L-arabinose
Week 6
04.08.2020
Assembled plasmids 12 & 13 by Gibson & transformation into Turbo and Nissle and plated into LBA + Amp100
05.08.2020
Colony PCR of the transformants found to confirm the plasmid
Sent plasmids 12 & 13 for sequencing to confirm the insertion of azurin
06.08.2020
Set up overnight colonies of plasmids 12 and 13 to transform into final strains
07.08.2020
Transformation of plasmids 12 and 13 into BL21 after sequencing confirmation
Week 7
10.08.2020
Transformations into BL21 unsuccessful
12.08.2020
Transformed plasmid 12 into Nissle and BL21
13.08.2020
Transformation of plasmid 12 into BL21 unsuccessful so started purification in Nissle
Transformed plasmid 11 into BL21 and Nissle for azurin expression and purification
14.08.2020
Small-scale purification of azurin from plasmid 12 in Nissle
Week 8
17.08.2020
Transformed plasmid 13 into Nissle
Inoculated LB + antibiotics with Nissle containing plasmid 10 or plasmid 11 for purification
18.08.2020
Attempted small-scale purification from plasmids 10 and 11 in Nissle
Also purified a negative control (pJ2060 + p41)
Ran results on 15% SDS-Page gel
Week 9
24.08.2020
Set up overnight cultures in LB + antibiotics for Nissle containing plasmid 10 (10mL) for large-scale purification and Nissle containing plasmid 13 for small-scale purification
25.08.2020
Started purification protocol for both plasmids (growth at 20°C)
1L used for large-scale purification of azurin from plasmid 10 in Nissle
Induced both after 4h growth with 0.2% L-arabinose
Gel of plasmid 10 didn’t give any clear bands where expected so most likely that azurin wasn’t being produced
Destained gel with both plasmid results overnight
Week 10
1.10.2020
Strains with the following plasmids coding for azurin were grown overnight at 37°C in LB:
Nissle pJ2060-N2only
Nissle p145-peIB-azu
Nissle p145-azu
Nissle p145-peIB-azu(ETHZ)
Nissle p145-azu(ETHZ)
BL21 pET22-azu
BL21 pET22-azu + IPTG
BL21 strain were induced the next day with 1mM IPTG for 4h at 37°C.
The OD of the cultures was measured to adjust the amount of cell culture to lyse
Ran results on 15% SDS-Page gel
No clear difference in bands present in p145-azu and induced BL21 pET22-azu plasmids and Nissle pJ2060-N2-only and uninduced BL21 pET22-azu
05.10.20
Growing the following strain until exponential phase:
Nissle p23
Nissle p24
Nissle p25
Nissle p26
Nissle p39
Nissle p40
BL21 pET22-azu1
BL21 p9
Nissle p41
200 uL of inoculation were put in 20 mL LB media and grown for 5 hours
pET22-azu1 and p9 are induced with 500uM IPTG at the same time
BL21 pET22-azu1 and BL21 p9 are also inoculated at 1% in 5mL LB with 50 or 500uM IPTG and grown at 20°C overnight
06.10.2020
Ran results on 15% SDS-Page gel
MTT Assay
We started our MTT assay about 4 weeks.
Week 4
27.07.2020
Reception of the Caco-2 cells and preparation to put them into Caco2 cells medium to let them grow
Week 6
07.08.2020
Check cell density and split the cells
Week 7
10.08.2020
Check cell density and make 4 stocks in freezing media (10% DMSO + 90% FBS)
11.08.2020
Prepare the dilutions of the treatments for the MTT assay
12.08.2020
Passage n°59 of the cells, according to subculture protocol, wash the cells with 1x Dulbecco’s PBS, and transfer the cells into new growth media
Week 8
17.08.2020
Passage n°60 of Caco-2 cells, wash the cells with 1x Dulbecco’s PBS, seed them and put them into 96-wells plate for the first MTT assay
20.08.2020
Set up the MTT assay. Treated the cells with the following different treatments:
Controls (DMSO or media or 0.1% Triton X or lysis buffer)
Different concentrations of Anisomycin: 50, 150, 300 and 450 [ng/ml]
Different concentrations of Cisplatin: 2.5, 5, 10 and 100 [μM]
Different concentration of Doxorubicin: 0.5, 1, 5 and 10 [μM]
Different concentration of Salirasib: 2.5, 5, 10 and 50 [μM]
Different concentration of TNFa: 1, 5, 10, 100 [ng/ml]
We did the same treatments for THP1 cells
Put to incubate at 37°C for 20h
21.08.2020
Continued the MTT assay, incubated 4h
Measuring absorbance at 570 nm using a plate reader
Week 9
26.08.2020
Redo the MTT assay for Caco-2 and THP1 cells with the same treatments but different conditions:
Controls (DMSO or media or 0.1% Triton X)
Different concentrations of Cisplatin: 10, 50 and 500 [μM]
Different concentration of Doxorubicin: 3, 10 and 100 [μM]
Different concentration of Salirasib: 1, 10 and 100 [μM]
Week 16
10.10.2020
O/n cultures in LB media with aTC and appropriated antibiotics with the following strains:
Nissle pLPT234 + pLPT145
Nissle pLPT234 + pLPT145-azu
Nissle pLPT234 + pLPT145-5D-azu.1_60del
Nissle pLPT234 + pLPT145-NSP4-azu.1_60del
The different strains above reached were diluted to an OD=0.5 before lysate the bacteria
Week 17
16.10.2020
Caco2 cells viability assay with bacterial supernatant and pellet lysate, plus with controls following the MTT assay protocol
Inoculation of Hela and HAP1 cells
Week 18
18.10.2020
O/n cultures in LB media with aTC and appropriated antibiotics with the following strains:
Nissle pLPT234 + pLPT145
Nissle pLPT234 + pLPT145-azu
Nissle pLPT234 + pLPT145-5D-azu.1_60del
Nissle pLPT234 + pLPT145-NSP4-azu.1_60del
19.10.2020
Lysate the bacteria above
20.10.2020
MTT assay: incubate Hela and HAP-1 cells bacteria supernant, pellet lysate and controls
Kill Switch
Week 9
25.08.2020
Assembly of pKA1-ccdB-ccdA
Tried with different polymerase (Q5, Vazyme and Phanta)
26.08.2020
Assembly of the different parts to design pAND-FRT-kan
Fragment
template
FW
REV
T_ann
t_elong
Amplicon size
concentration
260/280
260/230
Fmol/uL
FRT_1
pAND
PR104
PR105
65°C
40''
900bp
28 ng/uL
1.8
0.9
50
FRT_2A
pKA1 C7
PR103
PR106
55°C
4'
5kb
231 ng/uL
75
FRT_2C
pKC1 C12
PR103
PR106
55°C
4'
5kb
284 ng/L
90
PR103
pAND-FRT-rev
PR104
kanR-FRT-fw
PR105
kanR-FRT-rev
PR106
pAND-FRT-fw
Week 10
03.09.2020
Reamplification of pAND backbone (AB13) with Q5 and Phanta
With these amplicons, assembled by Gibson Assembly
Once the plasmid assembled, transformed into Nissle and Turbo
04.09.20
Colony PCR
07.09
Sequencing results
Week 11
15.09.20
The restreak of the colonies C8 and C12 showing no mutations in the sequencing did no grow back well
Reassemble the plasmids and plate the transformants on LB infused with aTc or IPTG (since the gene can be inserted under both promoters), so that the antitoxin is expressed and not killed by the antitoxin
The backbones fragments AB13 and AB14 were reamplified and digested using Dpn1
Week 12
23.09.2020
Reamplification of some fragments
Gibson Assembly of these fragments
Transformation
25.09.2020
Colony PCR
26.09.2020
Sequencing results good
Week 13
30.09.2020
Transformation into Nissle
A big thank you to our sponsors for their valuable support!
