A culture tube was prepared with 8 mL LB broth and 8 µL kanamycin. A small amount of the BL21 glycerol stock was inoculated into the culture tube. The small culture was incubated overnight at 37 °C at 200 rpm.
A culture flask with 250 mL of autoclaved TSB medium was prepared. 250 µL kanamycin was added. The 8 mL small culture of BL21 was inoculated into the culture flask and this large culture was incubated overnight at 37 °C at 200 rpm.
The large culture was pelleted at 6.000 x g for 20 min. at 4 °C. The supernatant was collected and filtered with a 0.2 µm pore size filter. The supernatant was filtered further with 100 kDa Amicon filters. The retentate was collected and analysed with Dynamic Light Scattering (DLS).
The prepared BL21 BMV sample was analysed with DLS. The number mean and intensity mean did not correspond to the expected size range for BMVs. Therefore, it was decided to look for an improved protocol.
Three culture tubes were made with 7.2 mL 7H9 Middlebrook and 800
µL ADC enrichment per tube. A small amount of
the M. smegmatis mc² 155 glycerol stock was inoculated into the culture
tube. To identify correct penicillin concentration, a different
concentration was added to each culture tube; 20 units/mL, 75
units/mL and 150 units/mL.
The small cultures were incubated overnight at 37
°C at 200 rpm.
Next to that, 2 L of Sauton's minimal medium was prepared and autoclaved.
The three small cultures of M. smegmatis were analysed with OD600 value. The OD600 of the small culture with 75 units/mL penicillin was comparable with the OD600 that is mentioned in literature. Therefore, this concentration was chosen as the standard penicillin concentration.
The small culture was inoculated into a large culture of 330 mL
Sauton's minimal medium with 75 units/mL penicillin. The OD600 was
measured every hour (Table 1).
| Time | OD600 |
|---|---|
| 11:10 | 0.001 |
| 14:10 | 0.010 |
| 15:10 | 0.012 |
| 16:10 | 0.006 |
| 17:10 | 0.011 |
The OD600 of the large culture was measured again. There was still no growth visible after 24 hours. Therefore, it was decided to redo the experiment with fresh Sauton's minimal medium.
Three small cultures of M. smegmatis in 7H9 Middlebrook with 75 units/mL penicillin were made and incubated overnight at at 37 °C at 200 rpm.
Small culture of M. smegmatis was inoculated in fresh Sauton's minimal medium large culture. OD600 was measured (Table 2).
| Time | OD600 |
|---|---|
| 10:00 | 0.056 |
| 12:30 | 0.067 |
| 13:45 | 0.003 |
| 15:30 | 0.000 |
| 16:15 | 0.001 |
Yeast extract was added in three different concentrations to the Sauton's minimal medium: 1 g/L, 5 g/L and 10 g/L. Next to that, a large culture with 7H9 Middlebrook and 10% ADC enrichment was tested. The OD600 was measured to monitor the growth (Table 3).
| Time | 7H9 Middlebrook with ADC | 1 g/L yeast extract | 5 g/L yeast extract | 10 g/L yeast extract |
|---|---|---|---|---|
| 9:30 | 0.041 | 0.056 | 0.055 | 0.047 |
| 10:45 | 0.040 | 0.043 | 0.035 | 0.037 |
| 12:00 | 0.047 | 0.051 | 0.040 | 0.046 |
| 13:50 | 0.073 | 0.058 | 0.040 | 0.040 |
| 15:20 | 0.096 | 0.062 | 0.061 | 0.061 |
| 9:00 - Next day | 0.774 | 2.017 | 1.503 | 0.620 |
| 10:15 | 0.893 | 2.212 | 1.721 | 0.702 |
As M. smegmatis grows best in Sauton's minimal medium with 1 g/L yeast extract, this was chosen as the large culture growth medium.
Three small cultures of M. smegmatis in 7H9 Middlebrook with 75 units/mL penicillin were made and incubated overnight at 37 °C at 200 rpm.
Small culture of M. smegmatis was inoculated in a large culture of 350 mL fresh Sauton's minimal medium with 1 g/L yeast extract and 75 units/mL penicillin. The starting OD600 of the large culture was 0.089. The large culture was incubated at 37 °C at 200 rpm. 4 hours later, the OD600 was 0.271. The large culture was incubated overnight.
The OD600 of the large culture was measured again and was 0.074. This suggested that the large culture did not survive. Next to that, floating particles were visible in the medium, which usually mean death cells. Therefore, a new large culture was made with Sauton's minimal medium with 1 g/L yeast extract and 75 units/mL penicillin. But this time, also 35 mL of ADC enrichment was added. The starting OD600 was 0.036. The large culture was incubated at 37 °C at 200 rpm overnight.
The OD600 of the large culture was measured again and was 1.756. The large culture was pelleted by centrifugation for 10 min. at 3.200 x g at RT. The cells were washed two times with Sauton's minimal medium. After washing, the cells were resuspended in Sauton's minimal medium and inoculated in three flasks with 315 mL Sauton's minimal medium, 1 g/L yeast extract, 35 mL ADC enrichment, 75 units/mL penicillin and 17.5 µL Tween-80 (0.005%). The cells were incubated overnight at 37 °C at 200 rpm and monitored the next day until OD600 reached ~2.0 to 2.5.
Large culture was centrifuged in three 400 mL centrifuge bottles at 4 °C for 20 min. at 8.000 x g. Supernatant was collected and filtered with 0.45 and 0.2 µm vacuum filters. After this, the filtered supernatant was concentrated with 30 kDa Amicon filters. The retentate was collected and a two-step centrifugation was done, first at 4.000 × g and then at 15.000 × g, both steps 4 °C for 20 min.
Half of the filtered sample was further concentrated via ultracentrifugation at 4 °C at 65.000 x g for 4 hours. After this, the pellet was resuspended in 50 mM HEPES buffer and stored in the fridge. The other half of the sample was stored for transport to Nijmegen.
Before transport of the sample to Nijmegen, a part of the sample was distributed over a 7H9 agar plate to confirm that no living bacteria were present in the sample anymore. This was grown in the incubator for around 3 days.
The 7H9 agar plate was checked for colonies. No colonies were visible, meaning that sample was safe for transportation out of the lab
The sample was packaged in a styrofoam box, filled with ice. This box was sealed with tape and placed in a plastic bag for convenient transportation. At the lab in Nijmegen, 14 mL of sample was ultracentrifuged at 4 °C at 100.000 x g for 4 hours. After this, the pellet was resuspended in 50 mM HEPES buffer and transported back to the lab in Eindhoven. On the next days, DLS measurements were performed on both samples.
Three small cultures of M. smegmatis in 7H9 Middlebrook with 75 units/mL penicillin were made and incubated overnight at 37 °C at 200 rpm.
Small culture of M. smegmatis was inoculated in a large culture of 315 mL fresh Sauton's minimal medium with 1 g/L yeast extract, 75 units/mL penicillin and 35 mL of ADC enrichment. The starting OD600 of the large culture was 0.035. The large culture was incubated at 37 °C at 200 rpm. The growth was monitored.
When OD600 reached ~1, the large culture was pelleted by centrifugation for 10 min. at 3.200 x g at RT. The cells were washed two times with Sauton's minimal medium. After washing, the cells were resuspended in Sauton's minimal medium and inoculated in two flasks with 315 mL Sauton's minimal medium, 1 g/L yeast extract, 35 mL ADC enrichment, 75 units/mL penicillin and 17.5 µL Tween-80 (0.005%). The cells were incubated over the weekend at 37 °C at 200 rpm.
Large culture was centrifuged in three 400 mL centrifuge bottles at 4 °C for 20 min. at 8.000 x g. Supernatant was collected and filtered with 0.45 and 0.2 µm vacuum filters. After this, the filtered supernatant was concentrated with 30 kDa Amicon filters. The retentate was collected and a two-step centrifugation was done, first at 4.000 × g and then at 15.000 × g, both steps 4 °C for 20 min.
After this, the filtered sample was further concentrated via ultracentrifugation at 4 °C at 65.000 x g for 4 hours. The pellet was resuspended in 50 mM HEPES buffer and stored in the fridge.
CryoTEM (Cryogenic Transmission Electron Microscopy) was performed to visualize the particles in the sample.
Furthermore, the sample was analyzed using DLS over the course of several days to monitor its size distribution and stability over time.
LB broth and LB-agar were prepared.
We prepared two kinds of agar plates. One with 160 mL LB agar and 160 µL of 50 mg/mL kanamycin (KAN), with a final concentration of 50 µg/mL and one with 320 mL LB agar and 640 µL of 50 mg/mL hygromycin B (HYG) with a final concentration of 100 µg/mL.
In preparation for the linearization PCR reaction of vector pET28a, the primers were diluted to 10 µM.
We linearized pET28a and then put the sample on an agarose gel. The gel confirmed that the pET28a linearization was successful.
We preformed a Gibson assembly on pET28a with (1a) OmpAN21-GFP, (1b) OmpA-GFP, (2a) CysSpycatcher and (2b) Spytag-GFP. After that the Gibson assembled plasmids were transformed into E. coli TOP10 cells by heat shock and plated onto LB agar with with 50 µg/mL KAN.
The TOP10 cells with the Gibson-assembled pET-28a plasmids resulted in colonies for inserts 1a, 2a and 2b but not for 1b. Therefore, three small cultures were started for 1a, three for 2a and two for 2b, using 8 mL of LB broth + 8 µL KAN (50 µg/mL).
The small cultures of TOP10 cells containing plasmids (1a) pET28a_OmpAN21-GFP, (2a) pET28a_CysSpycatcher and (2b) pET28a_Spytag-GFP were miniprepped and the concentrations were checked with the nanodrop.
Since the transformation of the Gibson assembled plasmid pET28a_OmpA-GFP (1b) did not result in any colonies, we decided to transfrom 1b into TOP10 cells again.
The miniprepped plasmids of (1a) pET28a_OmpAN21-GFP, (2a) pET28a_CysSpycatcher and (2b) pET28a_Spytag-GFP were sent for sequencing by Azenta. In the mean time, we put the miniprepped plasmids on an agarose gel to visualize our plasmids. The bands were at the expected places, meaning that we successfully created our desired plasmid.
The heat shock transformation of (1b) pET28a_OmpA-GFP did not result in colonies, so we retried the Gibson assembly and then transformed again into TOP10 cells. The Gibson Assembly was performed with the same reaction conditions as the first experiment. The miniprepped plasmids (1a) pET28a_OmpAN21-GFP, (2a) pET28a_CysSpycatcher and (2b) pET28a_Spytag-GFP were transformed into E. coli BL21 cells by heat shock for protein expression.
The (1a) pET28a_OmpAN21-GFP, (2a) pET28a_CysSpycatcher and (2b) pET28a_Spytag-GFP returned from Azenta, but the results were nonspecific, with each sample yielding more than one distinct sequence. We decided to return to the step of growing TOP10 small cultures from a colony. Since we only had colonies of 2a, we started small cultures from those and we transformed the Gibson assembly products of 1a and 2b into TOP10 cells via heat shock.
Transformations of (1a) pET28a_Ompa-GFP and (2b) pET28a_Spytag-GFP into TOP10 cells resulted in 2 colonies for (1a) and no colonies for (2b), therefore we transformed (2b) into TOP10 cells again.
The heat shock transformation of (1a) pET28a_OmpaN21-GFP and (2b) pET28a_Spytag-GFP into TOP10 cells resulted in colonies, so for each, small cultures were made.
Small cultures of (2a) pET28a_CysSpycatcher were made and glycerol stocks of the (1a) pET28a_OmpAN21-GFP and (2b) pET28a_Spytag-GFP small cultures were made.
Since the transformation of (1b) pET28a_OmpA-GFP failed again, we decided to linearize the pET28a plasmid before we did Gibson Assembly of (1b). After Gibson Assembly, the transformation was done into TOP10 cells heat shock.
For TOP10 cells containing (2a) pET28a_CysSpyCatcher, glycerol stocks were made. Now, the successful transformed small cultures of TOP10 cells containing plasmids (1a) OmpAN21-GFP, (2a) CysSpycatcher and (2b) Spytag-GFP were miniprepped and send to Azenta for sequencing.
The transformation of (1b) pET28a_OmpA-GFP finally resulted in a colony. We started a small culture with this colony.
The small culture of (1b) pET28a_OmpA-GFP was used to make glycerol stocks and miniprepped. Afterwards, an agarose gel was made to visualize the (1b) plasmid. The gel was deemed successful and (1b) was send to Azenta for sequencing. Then, 1b was transformed into BL21 cells by heat shock for protein expression.
The transformation of (1b) pET28a_OmpA-GFP into BL21 cells resulted in colonies.
The Azenta results of plasmids (1a) OmpAN21-GFP, (2a) CysSpycatcher and (2b) Spytag-GFP showed that the sequences were correct, so we started small cultures with the BL21 colonies. Although we were still waiting on the Azenta results of (1b), we already started small cultures with BL21 colonies of (1b) as well.
The OD600 was measured until the required value 0.6 was reached. Afterwards, the Ni-columns for protein purification were made.
The CysSpycatcher and Spytag-GFP proteins were isolated by iMAC purification. SDS-PAGE gels showed good results, both 2a and 2b were visible at the expected molecular weights. In total 11.28 mg (2a) CysSpycatcher and 2.92 mg (2b) Spytag-GFP was extracted.
Something went wrong with the sample preparation of 1b, so a new sample was made today. (1b) was sent to Azenta for sequencing. The results confirmed that we finally had the desired recombinant plasmid.
(1a) and (1b) are expressed in E. coli BL21 and prepared for Fluorescence-Activated Cell Sorting (FACS) to check expression. The next steps can be seen in the timelines below: OmpA(N21) and OmpA with GFP and OmpA(N21) and OmpA - Antibody staining.
(2a) and (2b) are used for the post-insertion method. The next steps can be seen in the notebook about Functionalization M. smegmatis.
A culture tube was prepared with 8 mL LB broth and 8 µL kanamycin. A small amount of the transformed BL21 glycerol stock was inoculated into the culture tube. The small culture was incubated overnight at 37 °C at 200 rpm.
Two culture flasks were prepared with 1L of 2YT growth medium. These flasks autoclaved and stored in the fridge.
A blank was prepared from the 2YT growth medium for OD600
measurements. The small cultures were inoculated into the large
culture flasks. These were incubated at 37 °C at
200 rpm. The growth was monitored until the culture reached a
OD600 of ~0.6.
The larges culture of OmpA and OmpA(N21) were induced with 100
µL of 1M IPTG.
The four large cultures were incubated overnight at 18 °C at 150 rpm.
10 mL of each culture flask was collected into seperate 15 mL falcon tubes. FACS measurements were performed.
A culture tube was prepared with 8 mL LB broth and 8 µL kanamycin. A small amount of the transformed BL21 glycerol stock was inoculated into the culture tube. The small culture was incubated overnight at 37 °C at 200 rpm.
Four culture flasks were prepared with 1L of 2YT growth medium. These flasks autoclaved and stored in the fridge.
A blank was prepared from the 2YT growth medium for OD600
measurements. The small cultures were inoculated into the large
culture flasks. These were incubated at 37 °C at
200 rpm. The growth was monitored until the culture reached a
OD600 of ~0.6.
One large culture of OmpA was induced with 100
µL of 1M IPTG at a OD600 of 0.889, one was not
induced for a negative control. One large culture of OmpA(N21) was
induced with 100 µL of 1M IPTG at a OD600 of
0.794, one was again not induced for a negative control.
The four large cultures were incubated overnight at 18 °C at 150 rpm.
15 mL of bacteria culture for each sample for the FACS measurement was collected. The cells were pelleted at 3.200 x g for 20 min. at °4 C. The pellet was washed twice with PBS enriched with 0.5% BSA. After this, the pellet was incubated with 125 µL of primary antibody at a concentration of 0.5 µg/mL in PBS (0.5% BSA) for 1 hour at room temperature.
After incubation with the primary antibody, the cells were washed twice. After this, they were incubated with secondary antibody at a concentration of 5 µg/ml in PBS (0.5% BSA) for 1 hour at room temperature.
After incubation with the primary antibody, the cells were washed twice and inoculated in 5 mL PBS (0.5% BSA). FACS measurements were performed.LB broth and LB-agar were prepared.
Prepared two kinds of agar plates. One with 160 mL LB agar and 160 µL of 50 mg/mL kanamycin (KAN), with a final concentration of 50 µg/mL and one with 320 mL LB agar and 640 µL of 50 mg/mL hygromycin B (HYG) with a final concentration of 100 µg/mL.
In preparation for the linearization PCR reaction of vector pCHERRY3, the primers were diluted to 10 µM.
dH5α cells containing pCHERRY3 had fully covered the agar plate, leaving no single isolated colony, so a touch of a pipet tip of the bacteria was resuspended in 1 mL of LB broth. This was diluted 1000x in LB broth and 100 µL was plated onto LB agar with 100 µg/mL HYG. Two such plates were made: one was incubated overnight at 37 °C and another one at room temperature (RT).
dH5α cells containing pCHERRY3 had formed single isolated colonies, so a small culture was inoculated (8 mL LB broth + 160 µL HYG) and incubated overnight at 250 rpm and 37 °C.
We realised we did not add the correct HYG concentration to the small culture of the dH5α cells containing pCHERRY3, so no bacteria had grown. A new small culutre was inoculated (8 mL LB broth + 16 µL HYG) at 100 µg/mL and incubated overnight at 250 rpm and 37 °C.
The small culture of dH5α cells containing pCHERRY3 was used to make glycerolstocks (550 µL culture + 450 µL glycerol). The small culture was also used to miniprep to isolate pCHERRY3.
We attempted to linearize pCHERRY3, then put the sample on gel to see whether the linearization was successful. The gel suggested that the linearisation was unsuccessful.
The lineazisation of pCHERRY3 was attempted again, but with a changed annealing temparature, namely from 65 °C to 67 °C. The agarose gel that we ran afterwards suggested that the linearization had failed again.
Linearization of pCHERRY3 plasmid was reattempted with different annealing temperatures, namely 55, 60 and 65 °C. The concentration of the pCHERRY3 samples were quite low, indicating that the linearization had failed again.
The linearization of pCHERRY3 was reattempted with an extension time of 420 s instead of 165 s (75 s/kb instead of 30 s/kb). This was performed at annealing temperatures 55, 60, 65, 66 and 67 °C and 34 cycles were performed instead of 32.
A new agarose gel was prepared to visualize the PCR products of the pCHERRY3 linearization with extension time 420 s and varying annealing temperatures (55, 60, 65, 66 and 67 °C). Only at annealing temperature 55 °C a band showed up, but not the correct one. We decided to retry the gel with the same samples to eliminate human error.
A new agarose gel was prepared to visualize the PCR products of the pCHERRY3 linearization with extension time 420 s and varying annealing temperatures (55, 60, 65, 66 and 67 °C). The band that was shown the second time was was a fat smear, so the linearization failed.
Since the band on the agarose gel resulted into a fat smear, we wanted to perform a restriction of the plasmid to check if it is the correct sequence. The plasmid was restricted by XbaI, PstI, HindIII and AgeI.
An agarose gel was made to visualize the result of the digestion of pCHERRY3 with PstI, SbaI, AgeI and HindIII. We only saw a smear of DNA fragments which indicated that we were using the incorrect plasmid. We decided to start a new culture of cells and do miniprep again.
We made small cultures with a higher HYG concentration than the previous time, one with 100 µg/mL and one small culture with 180 µg/mL.
The small cultures with the higher HYG concentration were much more red, suggesting that it contained more dH5α cells containing pCHERRY3.
We made a new dH5α with pCHERRY3 streak plate.
A new linearization of pCHERRY3 was performed with 34 PCR cycles instead of 32. On the streak plate of dH5α with pCHERRY3, many colonies with a pinkish color had appeared, indicating that they indeed contained pCHERRY3. Three small cultures were made from a single colony, with a hygromycin B concentration of 180 µg/mL.
The small cultures of dH5α with pCHERRY3 were miniprepped, resulting in three samples, then the concentration was measured with the nanodrop. Afterwards the miniprepped samples were put on an agarose gel. Next, the samples were send to Azenta for a sequencing check. With the new miniprepped samples, we retried linearization. To increase the chance of success, we varied two parameters: we used four annealing temperatures: 60, 63, 65 and 67 °C and we used two different template concentrations (0.01 and 0.02 ng/µL, while the protocol only indicates to use 0.01 ng/µL).
We put the samples on an agarose gel to analyze the results. However, there the correct band was not visible, indicating that the linearization had failed again, but that there was a contamination. Therefore, we decided to perform a gel extraction of the sample.
To even further increase the chance of a successful linearization, we performed a restriction first with PstI.
We made a new dH5α with pCHERRY3 streak plate, since we wanted to start fresh again. Small cultures was also started and was turning red which is in indication that the pCHERRY3 was present in the sample.
We miniprepped the new pCHERRY3 plasmid which was send to Azenta for sequencing.
The Azenta results came back confirming that we had the right plasmid sequence!
A new linearization was tried on the checked pCHERRY3 plasmid with an extension time of 210 s and an annealing temperature of 67°C.
An agarose gel was ran on the previously made samples, but to no luck. The wrong band was visible.
We used the supplied buffer in the PCR clean up process instead of nuclease free water, then ran an agarose gel again. But to no luck, the wrong band was visible again.
We tried a pCHERRY3 digestion again with the restriction enzymes: BamHI, PstI and HindIII. The samples were then loaded into an agarose gel to check whether the digestion was succesful, which it was! We decided to further linearize with PstI digested pCHERRY3 plasmid. We ran a PCR program that has been used before with an extension time of 210 s, with 33 cycles and an annealing temperature of 67 °C.
We ran an agarose gel to analyze our linearization. However, the gels showed no results.
It was suggested to us to try to linearize with a different protocol than the one we had used previously, namely switching from Phusion Master Mix to Q5. This time we prepared three samples: one with a double primer concentration, one adding a CG enhancer and both.
The agarose gel with the ran samples showed no result, indicating that the linearization had failed again. We decided to order new primers and try the linearization again.
The new primers arrived! We decided to mix the new and old primers to see whether that had any influence. We ended up with 4 new samples: one with new primers with the use of Q5, new primers with Phusion Master Mix, one with new forward primer and old reverse primer with Q5 and lastly the fourth sample with the old forward primer an the new reverse primer with Q5.
We visualized the four samples on an agarose gel, and the bands were still not at the right place, thus the linearization had failed again. We tried one more time with a longer extension time of 6 minutes in stead of 3.5 to see whether that had any influence. But the gel showed the wrong results once again. We decided to switch to restriction-ligation to create our recombinant plasmids.
While we were waiting for the new gBlock and primers, and since time was running out, we added our gBlock without first multiplying with PCR. So the concentration of the gBlock was a bit lower than we would have wanted. We only used PorinN50 (3c) for this and then thus did a plasmid digestion with pCHERRY3, (3c) and GFP for pCHERRY3.
We ran a gel on the digested pCHERRY3 plasmid to preform gel extraction and then performed the ligation and put the sample to react overnight in the fridge.
We transformed the made ligation into TOP10 cells, we added 8 µL of the sample instead of 2 µL, since the DNA concentration after the ligation was quite low.
On the plate two types of colonies were visible: one red (indicating the ligation had failed) and a white one. The white one could be our plasmid, so small cultures were made from the one colony to send to Azenta.
The small cultures that were made had grown, so we made glycerol stocks and miniprepped the samples to send to Azenta for sequencing.
While waiting for the Azenta results, we ran our sample on a gel to visualize the restriction ligation. The gel showed subtle indications that our plasmid was present, but it also revealed some contamination in the sample.
The Azenta results came back confirming that it was the wrong plasmid sequence.
We prepared a small culture by mixing 7H9 Middlebrook, ADC enrichment, and Penicillin, then inoculated it with M. Smegmatis glycerol stock. The culture was incubated overnight at 37 °C with shaking.
We set up a large culture by transferring 3 mL of the small culture into 300 mL of growth medium in a 1 L flask. This was incubated overnight at 37 °C until the OD 600 reached 0.8-1.0.
Cells were harvested by centrifugation, washed three times with ice-cold 10% glycerol, and resuspended in glycerol for storage at −70 °C or further use.
We performed the transformation of M. Smegmatis with pCHERRY3 using electroporation. The elecroporation machine was set to 1.8 ms and 1.5 kV. After that, we transferred the cell suspension to 7H9-ADCenr broth for a 2-hour incubation at 37 °C. The cells were then harvested by centrifugation and plated on 7H9-ADCenr-HYG agar, followed by incubation at 37 °C for 2-3 days to allow colony formation.
The transformation appeared to be succesfull, since the colonies were red. A small culture was made of M. Smegmatis with pCHERRY3 by inoculating 5 mL 7H9-ADCenr-HYG broth with transformant colonies, followed by incubation at 37 °C for 2-3 days.
We prepared three large cultures of M. smegmatis in 7H9 medium and measured the OD600 several times a day until it reached a value around OD600 0.80. We incubated overnight because the OD600 was too low to proceed.
After reaching an OD600 of 0.80, we added 0.1, 1, and 5 µg/mL doxycycline to the samples to assess which concentration led to the best protein expression. However, due to significant leak expression without doxycycline, inconsistent cell densities, and time constraints, we were unable to conclusively determine the optimal doxycycline concentration based on fluorescence.
For this method, the CysSpycatcher and Spytag-GFP proteins were expressed in E. coli BL21 and purified. For more information about this procedure, see the timeline for Cloning pET28a.
We incubated the purified proteins to check whether the Spycatcher and Spytag form a covalent isopeptide bond. The SDS Page gel confirmed that the coupling was successful.
Since we were unable to obtain DSPE in time, we made micelles using only DSPE-Mal to test if we could still functionalize the micelles with SpyCatcher. This experiment serves as a preliminary step to explore the possibility of future post-insertion with our BMVs. We began by generating a lipid film by dissolving DSPE-Mal in methylene chloride. After removing the solvent under a nitrogen stream, the lipid film was hydrated and sonicated to form micelles. CysSpyCatcher was then added to the micellar solution and incubated at room temperature for 1 hour to allow for functionalization.
After incubating the micelles with CysSpyCatcher, we used spin filtration to remove excess unreacted SpyCatcher. To check if micelles were formed, we performed DLS on samples containing either DSPE-Mal alone or DSPE-Mal coupled to CysSpyCatcher. We then tested functionalization by adding SpyTag-GFP to both CysSpyCatcher-conjugated micelles and non-functionalized micelles as a control. After filtering, we assessed fluorescence to determine if SpyCatcher-functionalized micelles successfully bound SpyTag-GFP.