iGEM 2024 — Team tu-eindhoven

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# **Notebook**

#### Write that down! Write that down!

##### *~ Spongebob Squarepants*

Take a look around the summary of our lab activities and full notebook!

---

BMV Isolation

Functionalization - *E. coli*

Functionalization - *M. smegmatis*

# *E. coli* BL21 BMV isolation

* June 16, 2024

  ### Small culture BL21

  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.
* June 17, 2024

  ### Large culture BL21

  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.
* June 18, 2024

  ### BL21 BMV isolation

  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).
* June 19, 2024

  ### BL21 BMV sample - DLS measurement

  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.

# *M. smegmatis* mc² 155 BMV isolation

* August 13, 2024

  ### Determining penicillin concentration

  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.
* August 15, 2024

  ### Large culture *M. smegmatis*

  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).

  *OD600 measurement of large culture of M. smegmatiswith
  Sauton's minimal medium.*

  | Time | OD600 |
  | 11:10 | 0.001 |
  | 14:10 | 0.010 |
  | 15:10 | 0.012 |
  | 16:10 | 0.006 |
  | 17:10 | 0.011 |

  Although there seemed to be no growth, the large culture was
  incubated overnight.
* August 16, 2024

  ### Large culture *M. smegmatis*

  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.
* August 23, 2024

  ### Small culture *M. smegmatis*

  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.
* August 26, 2024

  ### Large culture *M. smegmatis*

  Small culture of *M. smegmatis* was inoculated in fresh
  Sauton's minimal medium large culture. OD600 was measured (Table
  2).

  *OD600 measurement of large culture of M. smegmatis with
  Sauton's minimal medium.*

  | Time | OD600 |
  | 10:00 | 0.056 |
  | 12:30 | 0.067 |
  | 13:45 | 0.003 |
  | 15:30 | 0.000 |
  | 16:15 | 0.001 |

  It was concluded that Sauton's minimal medium is insufficient for
  *M. smegmatis* survival. Therefore, an improved minimal medium
  was made.
* August 27, 2024

  ### Improved Sauton's minimal medium

  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).

  *OD600 measurement of large culture of M. smegmatis with
  Sauton's minimal medium and different yeast extract
  concentrations*

  | 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.
* Augustus 30, 2024

  ### Small culture *M. smegmatis*

  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.
* September 2, 2024

  ### Large culture with improved Sauton's minimal medium

  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.
* September 3, 2024

  ### Addition of ADC enrichment to improved medium

  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.
* September 4, 2024

  ### Washing and inoculation of large culture

  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.
* September 5, 2024

  ### Filtering of large culture

  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.
* September 6, 2024

  ### Ultracentrifugation at 65,000 x g.

  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.
* September 10, 2024

  ### Ultracentrifugation at 100.000 x g.

  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.
* September 17, 2024

  ### Small culture of *M. smegmatis*

  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.
* September 19, 2024

  ### Large culture of *M. smegmatis*

  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.
* September 20, 2024

  ### Washing and inoculation of large culture

  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.
* September 23, 2024

  ### Ultracentrifugation at 65.000 x g

  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.
* September 24, 2024

  ### CryoTEM and DLS

  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.

# Cloning pET28a

* June 26, 2024

  ### Broth

  LB broth and LB-agar were prepared.
* June 27, 2024

  ### Agar plates

  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.
* July 1, 2024

  ### PCR preparation

  In preparation for the linearization PCR reaction of vector
  pET28a, the primers were diluted to 10 µM.
* July 8, 2024

  ### pET28a linearization

  We linearized pET28a and then put the sample on an agarose gel.
  The gel confirmed that the pET28a linearization was successful.
* July 9, 2024

  ### pET28a Gibson assembly

  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.
* July 10, 2024

  ### Gibson-assembled pET28a plasmids

  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).
* July 11, 2024

  ### TOP10 cells small culture

  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.
* July 12, 2024

  ### Gibson Assembled plasmid 1b

  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.
* July 15, 2024

  ### Miniprepped plasmids 1a, 2a and 2b

  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.
* July 16, 2024

  ### Transformation of 1b & Transformation into *E. coli* BL21 cells for 1a, 2a and 2b

  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.
* July 17, 2024

  ### Azenta results

  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.
* July 18, 2024

  ### Transformation of 1a and 2b

  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.
* July 19, 2024

  ### Small culture 1a and 1b

  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.
* July 22, 2024

  ### Small culture 2a & glycerol stocks 1a and 2b

  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.
* July 23, 2024

  ### Gibson Assembly 1b

  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.
* July 24, 2024

  ### Transformation 1b

  The transformation of (1b) pET28a\_OmpA-GFP finally resulted in a
  colony. We started a small culture with this colony.
* July 25, 2024

  ### Glycerol stocks 1b & Transformation 1b into BL21 cells

  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.
* July 26, 2024

  ### Sequence confirmation 1a, 2a, and 2b

  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.
* July 31, 2024

  ### Large culture 2a and 2b

  The OD600 was measured until the required value 0.6 was reached.
  Afterwards, the Ni-columns for protein purification were made.
* August 1, 2024

  ### Protein purification

  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.
* August 7, 2024

  ### Sequence confirmation 1b

  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***.

# OmpA(N21) and OmpA with GFP

* August 26, 2024

  ### Small culture of OmpA(N21) and OmpA

  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.
* Augustus 27, 2024

  ### Large culture OmpA(N21) and OmpA

  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.
* August 28, 2024

  ### Preparation FACS samples

  10 mL of each culture flask was collected into seperate 15 mL
  falcon tubes. FACS measurements were performed.

# OmpA(N21) and OmpA - Antibody staining

* September 17, 2024

  ### Small culture of OmpA(N21) and OmpA

  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.
* Septembr 18, 2024

  ### Large culture of OmpA(N21) and OmpA

  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.
* September 19, 2024

  ### Antibody staining

  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.

# Cloning pCHERRY3

* June 26, 2024

  ### Broth

  LB broth and LB-agar were prepared.
* June 27, 2024

  ### Agar plates

  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.
* July 1, 2024

  ### PCR preparation

  In preparation for the linearization PCR reaction of vector
  pCHERRY3, the primers were diluted to 10 µM.
* July 2, 2024

  ### dH5α cells colonies

  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).
* July 3, 2024

  ### dH5α cells colonies

  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.
* July 4, 2024

  ### PCR preparation

  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.
* July 5, 2024

  ### Small culture of dH5α cells

  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.
* July 8, 2024

  ### pCHERRY3 linearization

  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.
* July 9, 2024

  ### pCHERRY3 lineazisation

  The lineazisation of pCHERRY3 was attempted again, but with a
  changed annealing temperature, namely from 65 °C to 67 °C. The
  agarose gel that we ran afterwards suggested that the
  linearization had failed again.
* July 10, 2024

  ### pCHERRY3 linearization

  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.
* July 11, 2024

  ### pCHERRY3 linearization

  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.
* July 12, 2024

  ### pCHERRY3 linearization

  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.
* July 15, 2024

  ### pCHERRY3 linearization

  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.
* July 16, 2024

  ### pCHERRY3 Digestion

  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.
* July 17, 2024

  ### pCHERRY3 digestion

  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.
* July 18, 2024

  ### New dH5α cells colonies containing pCHERRY3

  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.
* July 19, 2024

  ### dH5α cells containing pCHERRY3

  The small cultures with the higher HYG concentration were much
  more red, suggesting that it contained more dH5α cells containing
  pCHERRY3.
* July 22, 2024

  ### New dH5α with pCHERRY3 streak plate

  We made a new dH5α with pCHERRY3 streak plate.
* July 23, 2024

  ### pCHERRY3 linearization

  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.
* July 24, 2024

  ### Small culture of dH5α with pCHERRY3

  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).
* July 25, 2024

  ### pCHERRY3 linearization

  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.
* July 26, 2024

  ### pCHERRY3 linearization

  To even further increase the chance of a successful linearization,
  we performed a restriction first with PstI.
* August 12, 2024

  ### New pCHERRY3 plasmid

  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.
* August 13, 2024

  ### New pCHERRY3 plasmid

  We miniprepped the new pCHERRY3 plasmid which was send to Azenta
  for sequencing.
* August 15, 2024

  ### Sequence confirmation pCHERRY3

  The Azenta results came back confirming that we had the right
  plasmid sequence!
* August 19, 2024

  ### pCHERRY3 linearization

  A new linearization was tried on the checked pCHERRY3 plasmid with
  an extension time of 210 s and an annealing temperature of 67°C.
* August 20, 2024

  ### pCHERRY3 linearization

  An agarose gel was ran on the previously made samples, but to no
  luck. The wrong band was visible.
* August 22, 2024

  ### pCHERRY3 linearization

  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.
* August 26, 2024

  ### pCHERRY3 digestion

  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.
* August 28, 2024

  ### pCHERRY3 linearization

  We ran an agarose gel to analyze our linearization. However, the
  gels showed no results.
* August 29, 2024

  ### pCHERRY3 linearization

  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.
* August 30, 2024

  ### pCHERRY3 linearization

  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.
* September 4, 2024

  ### pCHERRY3 linearization

  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.
* September 5, 2024

  ### pCHERRY3 linearization

  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.
* September 18, 2024

  ### pCHERRY3 restriction ligation

  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.
* September 19, 2024

  ### pCHERRY3 restriction ligation

  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.
* September 20, 2024

  ### pCHERRY3 restriction ligation

  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.
* September 23, 2024

  ### pCHERRY3 restriction ligation

  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.
* September 24, 2024

  ### pCHERRY3 restriction ligation

  The small cultures that were made had grown, so we made glycerol
  stocks and miniprepped the samples to send to Azenta for
  sequencing.
* September 25, 2024

  ### pCHERRY3 restriction ligation

  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.
* September 27, 2024

  ### Azenta result

  The Azenta results came back confirming that it was the wrong
  plasmid sequence.

# Transformation *M. Smegmatis*

* September 13, 2024

  ### Small culture

  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.
* September 16, 2024

  ### Large culture

  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.
* September 17, 2024

  ### Cell harvesting

  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.
* September 20, 2024

  ### Transformation

  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.
* September 23, 2024

  ### Small culture

  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.
* September 26, 2024

  ### Large culture

  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.
* September 27, 2024

  ### Doxycycline test

  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.

# Post-insertion

* August 2, 2024

  ### Preparation

  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.
* September 25, 2024

  ### Check CysSpycatcher and Spytag-GFP coupling

  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.
* September 26, 2024

  ### Micelle formation

  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.
* September 27, 2024

  ### Conjugation

  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.

Notebook Cloning and Protein Expression

{% endblock %}

{% extends "layout.html" %} {% block pagetitle %} Engineering Success {%
endblock %} {% block title %}{% endblock %} {% block lead %} {% endblock %} {%
block page\_content %}

# **Engineering**

#### Trust me, I'm an engineer

##### *~ TheVilkaz*

Creating something new is rarely a linear process. Regardless of planning
or research, many unexpected challenges arise. In our experiments we
planned for the unexpected by using the iGEM engineering cycles to build
from a minimal viable product to a final product. We also used this
approach in the modeling where we iteratively improved our model.

---

# Lab Engineering Cycle

### BMV Isolation engineering cycle

For the isolation of bacterial membrane vesicles (BMVs) from both*E. Coli*
and *M. Smegmatis*, multiple design cycles have been performed.

##### Cycle 1

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/build.svg)

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/design.svg)

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/learn.svg)

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/test.svg)

Click to see the next step

###### Design

Literature research resulted in a first draft version of a potential
protocol. This protocol can be found
[here](experiments#EColiIsolationProtocol). The protocol
was altered where necessary to fit our project.

###### Build

The protocol was performed in the lab.

###### Test

With the help of DLS the sample that resulted from the protocol was
analysed. The same sample was measured on day one and day five, both
for the charachteristics and the stability of the sample.

###### Learn

The number and intensity mean of the sample were lower than the
expected value for BMVs.
The PdI and intensity mean increased
significantly over five days, confirming that the sample is not
stable. The parts within the sample most likely form aggregates. To
confirm this conclusion, CryoTEM measurement was performed.

CryoTEM results showed BMVs of ranging sizes in the sample.
Next to that, the sample contained some protein aggregates. It was
therefore concluded to improve the protocol to decrease
heterogeneity.

###### Design

The insights of the results of the previous protocol in
combination with new literature research resulted in an improved
protocol. Within this new protocol, 0.45 and 0.2
µm filters were used to filter out whole
bacteria and bacterial debris. 30 kDa amicon filters were used to
further concentrate the sample. To overcome the issue of small
membrane vesicle in the sample, a new growth medium was
introduced: Sauton's minimal medium. This Sauton's medium should
provide an environment that favors BMVs production at the expense
of bacterial growth.

###### Build

The improved protocol was performed in the lab.

###### Test

The sample was again analysed with DLS. This time, the same sample
was measured multiple days from day one till day ten.

###### Learn

Both the number mean and intensity mean were within the expected
size range for BMVs. The PdI was also lower than in Cycle 1,
suggesting that the sample was more homogenous. The sample was
stable for seven days, which is expected for BMVs. As the number
mean and intensity differed, it was decided to do a CryoTEM
measurement to visualize the sample.

##### Cycle 2

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/learn.svg)

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/test.svg)

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/build.svg)

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/design.svg)

##### Cycle 3

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/build.svg)

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/design.svg)

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/learn.svg)

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/test.svg)

###### Design

Now that the DLS measurements suggested that the protocol was
successfull in isolating BMVs, a fresh sample was prepared via
performing the same protocol as in Cycle 2. The sample was
analysed with a CryoTEM measurement.

###### Build

The protocol from Cycle 2 was performed.

###### Test

The sample was analysed with CryoTEM. The diameter of the BMVs was
measured by hand to get an estimation of the average size.

###### Learn

The CryoTEM showed that the sample contained BMVs. The total
amount of BMVs was lower than in the protocol of Cycle 1. The size
of the BMVs, measured by hand, corresponded most to that of the
number mean. Besides BMVs, the sample still contained protein
aggregates. Next to that, ruptured BMVs were visible, most likely
caused by the centrifugal force. Further improvement of the
protocol is advised.

---

### Cloning engineering cycle

For the cloning of the genes encoding for the fusion proteins that
were to be expressed in *M. smegmatis*, several engineering
cycles were performed.

##### Cycle 1

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/build.svg)

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/design.svg)

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/learn.svg)

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/test.svg)

Click to see the next step

###### Design

In order to clone inserts 3a-3g into the plasmid pCHERRY3, by
Gibson assembly, primers were designed to perform a PCR
linearization of the pCHERRY3 plasmid. For all inserts gBlocks
were designed with Gibson overlaps at the ends. Since all fusion
proteins consist of two main domains, namely the membrane-anchor
into two Gblocks that share the same Gibson overlap in the middle.
The PCR program for the linearization of pCHERRY3 was designed as
shown in .

###### Build

The PCR linearization of pCHERRY3 was performed.

###### Test

The PCR product was run on an agarose gel.

###### Learn

No clear fragment was visible. We hypothesized that the annealing
temperature was too high for the primers to bind.

*PCR program for the linearization of pCHERRY3.*

| Stage | Temperature (°C) | Time |
| Initial denaturation | 98 | 60 s |
| Denaturation | 98 | 10 s |
| Annealing | 67 | 30 s |
| Extension | 72 | 165 s 32X |
| Final Extension | 72 | 5 min |
| End | 4 | Hold |

###### Design

The linearization PCR was redesigned with an annealing temperature
of 65°C instead of 67°C.

###### Build

The adjusted linearization PCR was performed.

###### Test

The PCR product was run on an agarose gel.

###### Learn

No clear fragment is visible. We hypothesized that the annealing
temperature is still too high for the primers to bind.

##### Cycle 2

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/learn.svg)

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/test.svg)

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/build.svg)

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/design.svg)

##### Cycle 3

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/build.svg)

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/design.svg)

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/learn.svg)

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/test.svg)

###### Design

The linearization PCR was redesigned with varying annealing
temperatures: 55°C, 60°C, 65°C, 66°C and 67°C.

###### Build

The adjusted linearization PCR was performed.

###### Test

The PCR product was run on an agarose gel.

###### Learn

At temperatures higher than 60°C, the agarose gel shows no clear
band. However, for 55 and 60°C, the agarose gel shows a fragment
at ~1.2 kb fragments instead of the expected ~5.6 kb fragment.
Therefore, we formulated and tested the three hypotheses seen in
.

* There is an alternative primer binding site for which annealing
  occurs at annealing temperatures of 60°C and below. We tested
  this hypothesis by analyzing the sequence of the plasmid for
  alternative binding sites that would result in a fragment of
  approximately 1.2 kb. We did not find such a primer binding
  site, only one that would result in a ~1.6 kb fragment. Because
  the difference between these fragments is quite small, we do not
  exclude the possibility that this hypothesis is true.
* The extension time of 165 seconds was insufficient for
  completing the replication of the full 5.6 KB fragment. In order
  to test this hypothesis we performed the linearization PCR with
  an extension time of 7 minutes. However, the same ~1.2 kb
  fragments were seen, proving this hypothesis incorrect.
* The sample of pCHERRY3 plasmid does not actually contain the
  plasmid. We tested this hypothesis by choosing a set of
  restriction enzymes that should cut the plasmids into a
  characteristic pattern of bands on the agarose gel. The result
  was merely a smear of DNA fragments that were smaller than
  expected. Therefore, we concluded that the linearization PCR of
  the plasmid was not working because the plasmid was incorrect.
  We remade the sample of the plasmid and confirmed its sequence
  by whole-plasmid-sequencing.

*Hypotheses resulting from Cycle 3*

###### Learn

No bands were seen on the gel. Therefore, we formulated the
following two hypotheses:
Linearizing the plasmid using a restriction enzyme prior to the
PCR, will enhance the efficacy of the replication.
The primers are malfunctioning

We decided to test the first hypothesis. We choose the PstI
restriction enzyme, which cuts within the sequence of the insert
that is not being copied by this linearization, see
. However, This strategy
once again resulted in no visible bands on the agarose gel,
making it seem more likely that the primers are the cause of the
problem.

###### Test

The PCR product was run on an agarose gel.

###### Build

The adjusted linearization PCR was performed.

###### Design

With the new sample of pCHERRY3 plasmid with the correct sequence,
we redesigned the linearization PCR, with varying annealing
temperatures of 55°C, 60°C, 65°C, 66°C and 67°C and with the
extension time of 7 minutes. To increase chance of success, we
performed the reactions with the Phusion Master Mix as well as
Q5-polymerase.

##### Cycle 4

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/learn.svg)

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/test.svg)

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/build.svg)

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/design.svg)

![pcher3 vector](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/pcherry.svg)

**Figure 1.**
*llustration of the restriction of the pCHERRY3 vector prior to
the linearization PCR.*

##### Cycle 5

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/build.svg)

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/design.svg)

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/learn.svg)

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/test.svg)

###### Design

We designed and ordered new primers to reattempt the linearization
PCR with varying annealing temperatures of 55°C, 60°C, 65°C, 66°C
and 67°C and with the extension time of 7 minutes. We did not only
reperform the linearization PCR with the new primers but also with
the combinations of a reverse primer from the old set and a
forward primer from the new set, and vice versa. Additionally,
since the plasmid is high in GC-content, we decided to also
perform a reaction with a GC-enhancer. We performed the reactions
with the Phusion Master Mix as well as Q5-polymerase.

###### Build

The adjusted linearization PCR was performed.

###### Test

The PCR product was run on an agarose gel.

###### Learn

No bands were seen on the gel. We decided to change the cloning
strategy from Gibson assembly to restriction ligation cloning.

###### Design

With the new sample of pCHERRY3 plasmid with the correct sequence,
we redesigned the linearization PCR, with varying annealing
temperatures of 55°C, 60°C, 65°C, 66°C and 67°C and with the
extension time of 7 minutes. To increase chance of success, we
performed the reactions with the Phusion Master Mix as well as
Q5-polymerase.

###### Build

Restriction ligation cloning was performed for fusion protein (3c)
PorinN50-GFP.

###### Test

The resulting plasmids were visualized on an agarose gel. Next,
the plasmids were transformed into TOP10 *E. coli* cells,
plated on selective media, cultured, miniprepped, and sent to
Azenta for sequence confirmation.

###### Learn

On the agarose gel of the plasmids with insert (3c) PorinN50-GFP,
bands are seen at the expected height (~6.5 kb), but other bands
are also seen at higher molecular weight. The gel can be viewed
[here](results#Agarosegel3cimg).

The Azenta results confirmed the sequence of the insert. This
still has to come true. In the next steps, we plan to clone the
plasmids with the inserts 3a, 3b and 3d-3g into pCHERRY3 by
restriction ligation cloning as well. For these plasmids, we
will also amplify the gBlocks prior to restriction ligation, to
increase the yield.

##### Cycle 6

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/learn.svg)

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/test.svg)

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/build.svg)

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/design.svg)

---

# Modeling Engineering Cycle

In parallel to the lab, the model also went through multiple cycles.
We worked in weekly sprints, where we would also look at the long term
planning and make adjustments to see which milestones were still
feasible. The modeling results inform the future steps of the lab in
future engineering cycles to be performed after the iGEM competition.
More details about the modeling can be found on the
[modeling](/model) page.

##### Cycle 1

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/build-model.svg)

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/design.svg)

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/learn.svg)

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/test.svg)

Click to see the next step

###### Design

We want to start with a preprocessed data file available publicly.
In this way we have results as fast as possible. We took some
inspiration from the
[Tongji China 2018](https://2018.igem.org/Team%3ATongji_China/Programme)
team for the intial idea but quickly went in another direction
after literature research.

###### Build

The main tool that we use, pVACtools, requires the data file to
have some annotations, so we implemented a step to add these.
Another input is required, the HLA type, which is available at the
data source.

###### Test

The main tool that we use, pVACtools, requires the data file to
have some annotations, so we implemented a step to add these.
Another input is required, the HLA type, which is available at the
data source.

###### Learn

We learn that other data sources may not provide an already
processed file and the HLA type.

###### Design

The goal is to not use the already processed data.

###### Build

We create an end-to-end pipeline that processes raw DNA data and
determines the HLA type. This involves learning about a lot of
bioinformatics tools, renting cloud computing, following tutorials
and bugfixing.

###### Test

We confirm that we get approximately the same results and that the
HLA type was correctly determined.

###### Learn

Neither the processed file or our pipeline is taking advantage of
all information, mainly RNA is not integrated

##### Cycle 2

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/learn.svg)

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/test.svg)

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/build-model.svg)

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/design.svg)

##### Cycle 3

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/build-model.svg)

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/design.svg)

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/learn.svg)

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/test.svg)

###### Design

The goal is to also integrate RNA information in the pipeline.

###### Build

We need to learn new tools but the concepts are often similar which
makes things faster. We upgrade our cloud computing because it is
taking a long time to run.

###### Test

We find that RNA information is now present in the output and that
the results changed.

###### Learn

We were already aware that our data had a limitation. It contained
only tumor DNA/RNA data and not a healthy control.

###### Design

After a long time we got access to a restricted dataset with a
healthy control. We want to analyse this data to see the effect

###### Build

We extend our pipeline to consider the healthy control. We also run
the original pipeline without the control.

###### Test

We check if the results of the tumor-only pipeline differs from the
pipeline with the control.

###### Learn

We find no differences for this particular patients but cannot draw
a general conclusion from this. We identify a way to reduce the need
for a healthy control, proteome similarity search, but will not have
time for another engineering cycle.

##### Cycle 4

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/learn.svg)

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/test.svg)

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/build-model.svg)

![](https://static.igem.wiki/teams/5403/wiki/engineering-cycle/design.svg)

{% endblock %}

{% extends "layout.html" %} {% block pagetitle %} Experiments {% endblock %}{%
block title %}{% endblock %} {% block lead %} {% endblock %} {% block
page\_content %}

# **Experiments**

#### It is only through failure and through experiment that we learn and grow.

##### *~ Isaac Stern*

Within this project, multiple experiments have been performed. To be able to
correctly perform all of our experiments, we made a clear protocol every
time. An overview of these protocols corresponding to the experiments can be
found on this page. Each protocol contains the goal of the protocol, the
materials used and the protocol itself. These protocols have also been
compiled into a single file. This file can be found at the bottom of the
page.

---

*E. Coli* Culturing and BMV Isolation

*M. Smegmatis* Culturing and BMV Isolation

Gibson Assembly cloning

Restriction-ligation cloning

Transformation of into TOP10 *E. coli* cells for plasmid
multiplication, purification and sequencing

Protein expression *E. coli*

Protein expression and antibody staining for FACS

SDS-PAGE

Transformation into *M. smegmatis* by electroporation

Determination of optimal doxycyclin concentration

Expression protocol in *M. smegmatis*

Media and agar plates for LB and 7H9

Post-insertion

All experiments of iGEM TU/e 2024

## Appendix

## References

Images created with BioRender.com

{% endblock %}

{% extends "layout.html" %} {% block pagetitle %} Results{% endblock %}{% block
title %}{% endblock %} {% block lead %} {% endblock %} {% block page\_content %}

# **Results**

#### I was taught that the way of progress was neither swift nor easy.

##### *~ Marie Skłodowska-Curie*

During the project, a wide variety of experiments were conducted, yielding
numerous results. These results were thoroughly analyzed, and conclusions
were drawn based on the findings. On this page, you will find detailed
information for each experiment, including the aim of the experiment, the
method used, the results, and the conclusions derived from the data. May
our findings guide your own experiments!

Content

×
[1. Achievements](#culture)
[2. BMV Isolation](#bmvisolation)
[3. Genetic Engineering](#geneticengineering)
[4. Post-insertion](#postinsertion)

---

# Achievements

### BMV Isolation

We successfully **isolated** BMVs from both
*E. coli* BL21 and *M. smegmatis* mc² 155, as can be
seen in the Cryogenic Transmission Electron Microscopy (CryoTEM)
visualizations of Figure 1 and Figure 2. We also succeeded in
increasing the **stability** of the BMVs by introducing
a HEPES buffer as a storage medium.

![CryoTEM results - BL21 BMV sample](https://static.igem.wiki/teams/5403/wiki/results/results-bmv-cryotem/bl51-e-coli-sample-1-2.jpg)

**Figure X:**  *CryoTEM of BL21 BMV sample.*

![CryoTEM results - M. Smegmatis BMV sample](https://static.igem.wiki/teams/5403/wiki/results/results-bmv-cryotem/cryotem-m-smegmatis-bmv-zonder-omcirkeling.webp)

**Figure X:**  *CryoTEM of M. smegmatis BMV sample.*

![DLS results - PdI of M. Smegmatis BMV sample](https://static.igem.wiki/teams/5403/wiki/results/results-bmv-dls/65k-protocol-pdi.jpg)

**Figure X:**  *PdI of M. smegmatis BMV sample.*

### Genetic Engineering

With the help of antibody staining, we were able to identify the
**fusion protein** using Fluorescence-Activated Cell
Sorting (FACS), consisting of the membrane protein OmpA(N21) fused
to GFP on *E. coli* BL21 cells. This proves successful membrane
functionalization of BL21 cells via genetic engineering. The FACS
result can be seen in Figure 4. The interpretation of the result can
be found [here](#FACSOmpA(N21)).

![FACS - OmpA(N21) + GFP antibody staining](https://static.igem.wiki/teams/5403/wiki/results/results-facs/1a/1a-p-s-signal.png)

**Figure X:** *FACS result of GFP fused to OmpA(N21) expressed in BL21 cells
(with IPTG and antibody staining).*

### Post-Insertion

For the post-insertion method, we successfully expressed and
purified the **CysSpycatcher** and
**Spytag-GFP** proteins from BL21 cells. Using
SDS-PAGE, we confirmed the formation of a covalent bond between
CysSpycatcher and Spytag-GFP (Figure 5). In a subsequent
experiment, we attempted to functionalize a DSPE-maleimide
(DSPE-mal) micellar solution with CysSpycatcher, preparing it for
future combination with BMVs for efficient functionalization.
While preliminary results suggest successful micelle formation and
functionalization with Spycatcher, further optimization is
required to improve homogeneity, consistency and stability.

![SDS page gel](https://static.igem.wiki/teams/5403/wiki/results/spycatcher-spytag.png)

**Figure X:** *SDS-PAGE gel showing successful covalent coupling between
CysSpycatcher (~10 kDa) and Spytag-GFP (~30 kDa). The
conjugated product (~40 kDa) is visible in the lanes
containing the mixture after incubation, confirming the
formation of the CysSpycatcher-Spytag-GFP complex.*

### Outlook

During the lab work and after the lab data analysis, we came up with
some future experiments that could guide the wet lab of our project into
the right direction. A detailed explanation of this lab outlook can be
found on the [Project Description page](description#Outlook).

# BMV Isolation

### Background

For BMV isolation, two protocols were used. The first protocol
focused on isolating BMVs from *E. coli* BL21 cells. The second
protocol, an improved version of the first protocol, focused on
isolating BMVs from *M. smegmatis* mc² 155. Below,
schematic representations of the protocols can be found.

Although our project focuses on *M. smegmatis*, we decided to
perform some of the lab work with *E. coli*. The reason for
this is both the faster growth of *E. coli* and the large
quantity of literature research on *E. coli*. These aspects
could increase accuracy and progression of the lab work.

Click to extend

![](https://static.igem.wiki/teams/5403/wiki/results/bmv-isolation-protocol-bl21.png)

**Figure X:** *Schematic representation of protocol for *E. coli* BL21
BMV isolation.*

![](https://static.igem.wiki/teams/5403/wiki/results/bmv-isolation-protocol-m-smegmatis-1.png)

**Figure X:** *Schematic representation of protocol for M. smegmatis BMV
isolation.*

![]()

### BMV isolation - *E. coli* BL21

*E. coli* BL21 cells were grown in Tryptic Soy Broth growth
medium to stimulate BMV formation. After that, the BMVs were
successfully isolated via multiple isolation and concentration
steps. The isolated BMVs were analysed with both DLS and CryoTEM.
With these results, an improved protocol was made to improve
specific parameters like homogeneity, vesicle size and protein
aggregate filtering.

##### Aim:

Stimulate BL21 cells to produce BMVs and isolation of these formed
BMVs.

##### Methods:

*E. coli* BL21 cells are capable of producing BMVs by
themselves. By filtering the bacterial culture with centrifugation
and Amicon filters, bacterial cells and debris can be removed from
the sample. This should result in a concentrated sample containing
BMVs. The protocol can be found
[here](experiments#EColiIsolationProtocol).
The concentrated sample was analyzed with DLS and CryoTEM. For DLS,
The sample was diluted in a 1:2 ratio with 30 µL sample and 30 µL
milli-Q water. The same sample was measured one day and five days
after completing the protocol. This was done to monitor the
stability of the sample. The expected size range of
*E. coli* BMVs is 50 – 200 nm
. The parameters used to analyze the DLS data were Polydispersity
Index (PdI), number mean and intensity mean.

**PdI** is a dimensionless number ranging from 0 to 1,
where 0 represents a monodisperse system, meaning all particles are
of the same size
. A
higher values indicate greater size heterogeneity.
**Number mean** is the average particle size calculated
by counting the number of particles in each size range. It
emphasizes the size of smaller particles because smaller particles
are typically more numerous. **Intensity mean** is the
average particle size based on the intensity of scattered light,
which tends to emphasize larger particles because the intensity of
light scattering increases with the sixth power of the particle
radius.

##### Results:

![DLS result - PdI of BL21 BMV sample](https://static.igem.wiki/teams/5403/wiki/results/results-bmv-dls/protocol-1-pdi.jpg)

**Figure X:**  *PdI of BL21 BMV sample.*

![DLS result - Number mean of BL21 BMV sample](https://static.igem.wiki/teams/5403/wiki/results/results-bmv-dls/protocol-1-number-mean.jpg)

**Figure X:**  *Number mean of BL21 BMV sample.*

![DLS result - Intensity mean of BL21 BMV sample](https://static.igem.wiki/teams/5403/wiki/results/results-bmv-dls/protocol-1-intensity-mean.jpg)

**Figure x:**  *Intensity mean of BL21 BMV sample.*

As seen in
, the PdI increases significantly over 5 days. This suggests that
the sample becomes more heterogenous over time, which could be due
to aggregates being formed or vesicle fusion. This increase in PdI
corresponds with the behavior of an unstable sample. As seen in
, the number mean is around 18 nm. This size range is unexpected for
*E. coli* BMVs, suggesting that the majority of the sample does
not consist of BMVs. Meanwhile, the number mean seems to be stable
over 5 days.
shows the intensity mean over 5 days. The intensity mean is
significantly higher than the number mean, suggesting that the
sample contains some large particles. Next to that, the intensity
mean of the sample increases significantly, suggesting an increase
in larger particles within the sample. This confirms the assumption
that the sample is not stable over time, as suggested with the
increased PdI.

To confirm the hypothesis that the sample most likely does not
contain
*E. coli* BMVs, a CryoTEM measurement was done.

![CryoTEM results - BL21 BMV sample](https://static.igem.wiki/teams/5403/wiki/results/results-bmv-cryotem/bl51-e-coli-sample-1-1.jpg)

**Figure X:**  *CryoTEM of BL21 BMV sample.*

![CryoTEM results - BL21 BMV sample](https://static.igem.wiki/teams/5403/wiki/results/results-bmv-cryotem/bl51-e-coli-sample-1-2.jpg)

**Figure X:**  *CryoTEM of BL21 BMV sample.*

Due to an error with the CryoTEM, the zoom capacity was limited to
a maximum of 500 nm. As we would expect the diameter of most
particles to be around 18 nm, it is difficult to analyze these
results. But by zooming in by hand, it is still possible to
identify some potential BMVs.

![CryoTEM results - BL21 BMV sample](https://static.igem.wiki/teams/5403/wiki/results/results-bmv-cryotem/bl51-e-coli-sample-1-1-1.jpg)

**Figure X:**  *Zoomed in image of CryoTEM of BL21 BMV sample.*

![CryoTEM results - BL21 BMV sample](https://static.igem.wiki/teams/5403/wiki/results/results-bmv-cryotem/bl51-e-coli-sample-1-1-2.jpg)

**Figure X:**  *Zoomed in image of CryoTEM of BL21 BMV sample.*

![CryoTEM results - BL21 BMV sample](https://static.igem.wiki/teams/5403/wiki/results/results-bmv-cryotem/bl51-e-coli-sample-1-2-1.jpg)

**Figure X:**  *Zoomed in image of CryoTEM of BL21 BMV sample.*

The sample contains some protein aggregates and debris, but most
abundant are the vesicles with a lamellar structure. These
vesicles show a wide range of sizes. In the largest vesicles
visible in the red squares (Figure 13, Figure 14 and Figure 15),
the lamellar structure can be observed. Although the overall
size of the vesicles is very small, this CryoTEM visualization
suggests that the isolation of BMVs may have been successful for
BL21 cells.

##### Conclusion:

The CryoTEM results show that the sample contains particles that
closely resemble vesicles. The size of these vesicles in the
sample differs and without zooming in correctly it is difficult to
distinguish between vesicles and protein aggregates. Next to that,
the size of the vesicles is smaller than expected, confirmed by
the number mean and CryoTEM.
The PdI and intensity mean show that the particles are not stable
over time.

The results suggest that the performed protocol can be improved.
The sample must be filtered more thoroughly to remove debris and
increase homogeneity. The isolated vesicles must be stored in a
stable buffer to retain their structual integrity.

### BMV Isolation - *M. smegmatis*

An improved protocol with respect to the BL21 BMV isolation was
performed on *M. smegmatis*. The protocol can be found
[here](experiments#MsmegIsolationProtocol). A new growth
medium, Sauton's minimal medium, was used to promote BMV production
of *M. smegmatis*. More filtration and concentration steps were
introduced by (ultra)centrifugation and by using 30kDa Amicon
filters. After isolation, DLS measurements were performed on the
sample to identify the homogeneity, size and stability of the
particles. The sample was stable for at least seven days, an
increase compared to the old protocol. Next to that, the number mean
was within the expected size range for *M. smegmatis* BMVs
. The homogeneity, represented by the PdI, was roughly the same as
the old protocol.

##### Aim:

Stimulate *M. smegmatis* cells to produce BMVs and evaluate
which isolation steps are necessary in an improved protocol.

##### Methods:

Literature research was performed to find an improved protocol for
isolation of BMVs. We looked for an isolation protocol that is
compatible to both *E. coli* and *M. smegmatis* for
comparison with respect to the old protocol and flexibility in
negative controls.

The improved protocol uses a minimal medium, two more filtration
steps via (ultra)centrifugation and the use of a smaller Amicon
filter of 30 kDa. The sample is, after isolation, stored in a 50 mM
HEPES buffer. These improvements should, in theory, result in a more
homogenous, stable sample.

To identify the effect of an ultracentrifugation step for
purification, the concentrated material is divided into two samples.
The first sample, 65K, is concentrated with ultracentrifugation at
65.000 x g, while the second sample, 100K, is concentrated with
ultracentrifugation at 100.000 x g.

##### Results:

![DLS result - PdI of M. Smegmatis BMV sample](https://static.igem.wiki/teams/5403/wiki/results/results-bmv-dls/protocol-2-pdi-1.jpg)

**Figure X:**  *PdI of M. smegmatis BMV sample.*

![DLS result - Number mean of M. Smegmatis BMV sample](https://static.igem.wiki/teams/5403/wiki/results/results-bmv-dls/protocol-2-number-mean-1.jpg)

**Figure X:**  *Number mean of M. smegmatis BMV sample.*

![DLS result - Intensity mean of M. Smegmatis BMV sample](https://static.igem.wiki/teams/5403/wiki/results/results-bmv-dls/protocol-2-intensity-mean-1.jpg)

**Figure X:**  *Intensity mean of M. smegmatis BMV sample.*

The DLS measurement, visible in
,
and
, starts at day 3, which is 3 days after finishing the isolation
protocol with the 65.000 x g ultracentrifugation step. The DLS
measurement for the 100K sample starts at day 5, as this protocol
took 2 days longer due to transportation to another lab. The PdI
of the 65K sample is stable for at least 7 days around 0.17, as
can be seen in
. The PdI of the 100K sample seems to fluctuate more, increasing
and decreasing over time. For both samples, the PdI is within the
acceptable range (<0.3) for a homogenous sample, for at least 7
days.
At day 10, the sample is not stable anymore, as the PdI increases
significantly.

shows that the number mean for both samples is within the expected
size range for BMVs (50-250 nm). On top of that, the number mean of the 65K sample does not
fluctuate significantly in 10 days.

The intensity mean of both samples does not fluctuate
significantly for at least 7 days, as can be seen in
. On day 4, the intensity mean of the 65K sample seems to be
higher than expected, with a relatively large error margin, which
may indicate a potential measurement error.
The intensity mean increases significantly on day 10 for both
samples, suggesting the increase of particle size within the
sample. This could be due to aggregates being formed or fusion of
vesicles.

##### Conclusion:

The DLS measurements suggests that the 65K sample is more stable
than the 100K sample, given by the PdI. The number mean and
intensity mean do not differ significantly between both samples.
This suggests that a 65.000 x g ultracentrifugation step is
preferred over a 100.000 x g step.

Compared to the sample in the BL21 protocol, the stability of the
this sample improved when stored in HEPES buffer. It is therefore
recommended to use a HEPES buffer for BMV storage.

Since the results are promising, the protocol will be repeated and
both DLS and CryoTEM measurements will be performed.

### BMV Isolation - 65.000 x g - *M. smegmatis*

Since the DLS measurements after the improved BMV isolation protocol
for *M. smegmatis* showed promising results, the protocol was
repeated and, next to a DLS measurement, a CryoTEM measurement was
performed to visualize the sample. The DLS measurement showed little
to no difference in number mean and intensity mean, but the PdI was
significantly smaller.

The CryoTEM showed BMVs, although a lower quantity than obtained
after the BL21 BMV isolation. The size of the BMVs was more
consistent. The sample still contained protein aggregates and some
of the BMVs were ruptured.

##### Aim:

Perform the protocol to isolate BMVs from *M. smegmatis* and
analyse the sample with both DLS and CryoTEM.

##### Methods:

Within the BMV isolation protocol for *M. smegmatis*, 65.000 x
g ultracentrifugation was chosen as this yielded better results than
100.000 x g ultracentrifugation. Apart from that, the protocol was
not altered and replicated from previous experiments.

##### Results:

![DLS result - PdI of M. Smegmatis BMV sample](https://static.igem.wiki/teams/5403/wiki/results/results-bmv-dls/65k-protocol-pdi.jpg)

**Figure X:**  *PdI of M. smegmatis BMV sample.*

![DLS result - Number mean of M. Smegmatis BMV sample](https://static.igem.wiki/teams/5403/wiki/results/results-bmv-dls/65k-protocol-number-mean.jpg)

**Figure X:**  *Number mean of M. smegmatis BMV sample.*

![DLS result - Intensity mean of M. Smegmatis BMV sample](https://static.igem.wiki/teams/5403/wiki/results/results-bmv-dls/65k-protocol-intensity-mean.jpg)

**Figure X:**  *Intensity mean of M. smegmatis BMV sample.*

The PdI, visible in
, is lower than in the previous experiment, around 0.10. This
suggests that the sample is very homogenous. For at least 7 days,
the PdI stays stable, suggesting good stability of the sample. In
, the number mean is very stable with little to no variation in
size. The same goes for the intensity mean in
. These results suggest that the sample shows the same behavior as
the previous sample, if not better. Therefore, CryoTEM is
performed to visualize the sample.

![CryoTEM result - M. Smegmatis BMV sample](https://static.igem.wiki/teams/5403/wiki/results/results-bmv-cryotem/cryotem-m-smegmatis-bmv.webp)

**Figure X:**  *CryoTEM of M. smegmatis BMV sample.*

![CryoTEM result - M. Smegmatis BMV sample](https://static.igem.wiki/teams/5403/wiki/results/results-bmv-cryotem/cryotem-m-smegmatis-ruptured-bmv.webp)

**Figure X:**  *CryoTEM of M. smegmatis BMV sample.*

![CryoTEM result - M. Smegmatis BMV sample](https://static.igem.wiki/teams/5403/wiki/results/results-bmv-cryotem/cryotem-m-smegmatis-protein-aggregates.webp)

**Figure X:**  *CryoTEM of M. smegmatis BMV sample.*

Within the red squares of
, BMVs are visible. These vesicles that are visible in CryoTEM are
in the same size range as the number mean measured by DLS.
Overall, the amount of BMVs seems to be lower than in the CryoTEM
results of BL21 BMV isolation, but the BMVs themselves are more
consistent in size.
shows lamillar structures, but they seem to have ruptured. This
could be due to the high forces on the vesicles during
ultracentrifugation or osmotic stress. In
, protein aggregates are visible. It can therefore be concluded
that with this protocol, we did not filter all the protein
aggregates in the sample.

##### Overall Conclusion:

In previous experiment, the BMV isolation of BL21 was successful,
shown by the CryoTEM results. But the BMVs are heterogenous and
their average size is smaller than expected. By storing the BMVs in
a HEPES buffer, which was done in the improved protocol, the
stability of the BMVs increased, resulting in the possibility for
long-term storage. The extra filtration and isolation steps in the
improved protocol resulted in a more homogenous BMV sample, but the
high forces, due to (ultra)centrifugation, most likely caused the
presence ruptured vesicles within the population of BMVs.

It is difficult to compare the two CryoTEM visualizations of (*E. coli*
BL21 and *M. smegmatis*) with each other, as two variables were
introduced: change in species and change in protocol. It is
therefore adviced to redo both experiments, in which both species
are analysed in parallel. This enhances the reliability of result
comparisons, leading to more informed decisions regarding the
optimal BMV isolation protocol.

# Genetic Engineering

### Background

Two strategies for the functionalization of the bacterial membrane
vesicles were investigated in the laboratory. The first strategy
to be discussed is the genetic engineering strategy, where fusion
proteins are used.

![Genetic engineering](https://static.igem.wiki/teams/5403/wiki/results/genetic-engineering2.png)

**Figure 1:** *Genetic engineering method: bacterial cell is transformed
with desired insert, leading to expression of a fusion
protein that includes the antigen (or model-antigen, GFP).
BMVs are isolated after fusion protein expression.*

In  the
genetic engineering method is visualized. Literature research was
performed to find proteins that are known to reside in the
membrane of the vesicles of mycobacteria (*M. smegmatis*,
*M. tuberculosis* and *M. bovis* BCG), that are suitable
for the attachment of model-antigen GFP at the periplasmic face of
the membrane. The following selection of such proteins was made:
OmpAtb, porin MspA, FtsQ, and RbsC. The structure of these
proteins was analyzed to identify certain domains or signal
sequences that can be used for the expression of a fusion protein
with GFP at its C-terminal end. It is important that the membrane
proteins retain their functional domains, to enable effective
display of the GFP on the outside of the BMV. Therefore, the
membrane proteins that are fused to the antigen are either:

* Full membrane proteins
* Truncated membrane proteins: consisting of the signal peptide
  and one or more transmembrane domains
* Truncated membrane proteins: consisting of the signal peptide
  only

For *M. smegmatis* several modeling tools were used to design
relevant fusion protein constructs, like DeepTMHMM, Signal6.0 and
Alphafold. More information can be found on our
[Modeling page](https://2024.igem.wiki/tu-eindhoven/model). This resulted in a selection of seven fusion proteins, labeled
3a-3g, described below:

* (3a) The 72 N-terminal amino acids of OmpAtb (signal sequence)
  with a flexible linker (GSGSGSGSAS) and GFP at its C-terminus
  (OmpAtbN72-GFP). Because of the virulent functions of the
  protein in M. tuberculosis, only this signal sequence is
  permitted for use in an ML-1 laboratory, instead of the full
  protein
* (3b) The 72 N-terminal amino acids of OmpAtb (signal sequence),
  with OmpA (the *E. coli* analog of OmpAtb), a flexible
  linker and GFP at its C-terminus (OmpAtb-OmpA-GFP)
* (3c) The 50 N-terminal amino acids of porin MspA, with a
  flexible linker and GFP at its C-terminus (PorinN50-GFP)
* (3d) Porin MspA without the 13 C-terminal amino acids, with a
  flexible linker and GFP at its C-terminus (PorinTrunc13-GFP)
* (3e) The full Porin MspA protein with a flexible linker and GFP
  at its C-terminus (Porin-GFP)
* (3f) RbsC missing 35 C-terminal amino acids with a flexible
  linker GFP at its C-terminus (RbsC-GFP)
* (3g) FtsQ with a flexible linker and GFP at its C-terminus
  (FtsQ-GFP)
* (3h) GFP (as control, to show that it can be expressed in
  *M. smegmatis*)

Apart from the fusion proteins in *M. smegmatis*, two fusion
proteins were designed for *E. coli*. This has been done in
parallel to be able to develop a proof of concept for
*E. coli* that is comparable for *M. smegmatis*. The
fusion proteins are based on outer membrane protein A (OmpA), a
membrane protein that is native to *E. coli*. The two fusion
proteins are:

* (1a) The 21 N-terminal amino acids of OmpA (signal sequence)
  with at its C-terminus flexible linker and GFP
* (1b) The full OmpA protein with at its C-terminus a flexible
  linker and GFP

An overview of the inserts, plasmids and hosts can be found in
Table 1. Inserts 1a and 1b are used for expression in
*E. coli* and 3a-3h for expression in *M. smegmatis*.
Inserts 2a and 2b are used for the post-insertion method, that
will be employed as an alternative for the genetic engineering
method. More information about 2a and 2b can be found in the
post-insertion section.

**Table 1:**
*Overview of proteins used for functionalizing BMVs. The table
lists the different protein inserts cloned into various
plasmid backbones, and their corresponding host organisms.*

| ID | Protein inserts | ID (parts registry) | Plasmid | Host |
| --- | --- | --- | --- | --- |
| 1a | OmpA(N21) + GFP | BBa\_K5403012 | pET28a | *E. coli* |
| 1b | OmpA + GFP | BBa\_K5403013 | pET28a | *E. coli* |
| 2a | CysSpycatcher + 6H | BBa\_K5403005 | pET28a | *E. coli* |
| 2b | Spytag + GFP + 6H | BBa\_K5403021 | pET28a | *E. coli* |
| 3a | OmpAtb(N72) + GFP | BBa\_K5403014 | pCHERRY3 | *M. smegmatis* |
| 3b | OmpAtb(N72) + OmpA + GFP | BBa\_K5403015 | pCHERRY3 | *M. smegmatis* |
| 3c | Porin(N50) + GFP | BBa\_K5403016 | pCHERRY3 | *M. smegmatis* |
| 3d | Porin (C-truncated) + GFP | BBa\_K5403017 | pCHERRY3 | *M. smegmatis* |
| 3e | Porin + GFP | BBa\_K5403018 | pCHERRY3 | *M. smegmatis* |
| 3f | RbsC (C-truncated) + GFP | BBa\_K5403019 | pCHERRY3 | *M. smegmatis* |
| 3g | FtsQ + GFP | BBa\_K5403020 | pCHERRY3 | *M. smegmatis* |
| 3h | GFP | BBa\_E0040 | pCHERRY3 | *M. smegmatis* |

### Cloning for *E. coli* proteins

The genes encoding the fusion proteins OmpA(N21)-GFP and OmpA-GFP
that are used for the genetic engineering method were cloned into
the pET28a vector by linearization and Gibson assembly.
CysSpycatcher and Spytag-GFP, that are later used for the
post-insertion method were cloned using the same approach.

##### Aim:

Linearization and Gibson assembly of fusion proteins 1a, 1b, 2a
and 2b into the pET28a plasmid.

##### Methods:

Vector linearization was achieved through PCR, followed by DpnI
digestion and a PCR purification step. The concentration was then
determined using NanoDrop. Subsequently, Gibson Assembly was
performed, and the resulting samples were visualized on an agarose
gel to check whether the correct plasmid was assembled.

##### Results:

![agarose gel 1a, 2a en 2b](https://static.igem.wiki/teams/5403/wiki/results/agarose-gel-met-1a-2a-en-2b.png)

**Figure x:** *Agarose gel showing the constructed pET28a plasmids
containing the genes encoding for the proteins (1a)
OmpA(N21)-GFP, (2a) CysSpycatcher and (2b) Spytag-GFP*

![agarose gel 1b](https://static.igem.wiki/teams/5403/wiki/results/agarose-gel-met-1b.jpg)

**Figure x:** *Agarose gel showing the constructed pET28a plasmid
containing the genes encoding for the protein (1b)
OmpA-GFP and the linearized pET28a vector*

and
 show the
agarose gels for the *E. coli* inserts. Afterwards, the
plasmids were sent to Azenta for sequencing.

##### Conclusion:

In
 and
, all the
plasmids are visible at their expected molecular weight. The
Azenta sequencing results confirmed this observation. So, we
succesfully assembled our plasmid.

### FACS - OmpA(N21) + GFP

The OmpA(N21) membrane protein, fused to GFP, is expressed in BL21
cells by induction of IPTG. The sample is stained with a secondary
antibody conjugated to a dye, to increase the fluorescent signal
of the GFP. The fluorescent signal is analysed with FACS.

##### Aim:

Analyze the presence of GFP, bound to membrane protein OmpA(N21),
on *E. coli* BL21 cells.

##### Methods:

To analyze the expression of both membrane proteins, a large
culture was made, consisting of 2YT medium with BL21 cells
transformed with a pET28a recombinant plasmid. The culture was
grown until a OD600 of ~0.6 was reached. At that point, IPTG was
added to the culture to induce protein expression and the large
culture was incubated at a lower temperature of 18 °
overnight. The protocol can be found
[here](experiments#FACSprotocol).

In the first setup of this experiment, the IPTG incubated large
culture was directly analyzed with FACS. These FACS results showed
little to no GFP signal. The experiment was repeated, but still no
signal was visible. To overcome this issue, an antibody staining
was introduced to increase the signal.

Antibody staining was done with a primary antibody for GFP and a
secondary antibody conjugated to a Cyanine3 dye. Several negative
controls were incorporated into the experiment:

* Negative control without IPTG induction. Used to conclude that
  the protein is not expressed when IPTG is not present.
* Negative control without antibody staining. Used to conclude
  that antibody staining is necessary for visualization of the GFP
  signal.
* Negative control without IPTG induction and without antibody
  staining. Used to determine a threshold for the signal noise.
* Negative control with only secondary antibody incubation. Used
  to analyze the occurence of non-specific staining.

##### Results:

![FACS result - Sample OmpA(N21) gated](https://static.igem.wiki/teams/5403/wiki/results/results-facs/1a/1a-unstained-cell-gated-facs.png)

**Figure X:**  *FACS result - Sample OmpA(N21) gated.*

![FACS result - Sample OmpA(N21) Single cells](https://static.igem.wiki/teams/5403/wiki/results/results-facs/1a/1a-unstained-single-cells-facs.png)

**Figure X:**  *FACS result - Sample OmpA(N21) single cells.*

![FACS result - Sample OmpA(N21) signal](https://static.igem.wiki/teams/5403/wiki/results/results-facs/1a/1a-unstained-signal-facs.png)

**Figure x:**  *FACS result - Sample OmpA(N21) signal.*

To analyze FACS results, the data has to be gated by hand. By
gating, unwanted data that would interfere with the analysis is
excluded. This reduces noise and improves data accuracy. Gating
is done based on the forward and side scatter of the sample. In
, the gating is shown.

To improve accuracy, doublet discrimination is used. Doublets
are events where two cells pass through the flow cytometer's
laser at the same time or very closely together, producing an
artifact in the data. These doublets can be mistakenly counted
as a single event, leading to inaccurate results. This
discrimination is done by comparing pulse height (FSC-H) and
pulse area (FSC-A) in
.

When both gating and doublet discrimination have been completed,
the quartiles lines that seperate the quartiles can be
determined. It is adviced that, in a negative control, maximum
1% of our signal falls in Q1 and Q2.
shows the correct separation.

Now that the FACS data is completely filtered, the settings for
the gate, doublet discrimination and placement of the quartile
lines can be repeated on all samples for OmpA(N21).

![FACS result - Sample OmpA(N21) signal](https://static.igem.wiki/teams/5403/wiki/results/results-facs/1a/1a-nc-unstained-signal.png)

**Figure X:** *FACS result of OmpA(N21) (no IPTG and no antibody
staining).*

![FACS result - Sample OmpA(N21) signal](https://static.igem.wiki/teams/5403/wiki/results/results-facs/1a/1a-unstained-signal-facs.png)

**Figure X:** *FACS result of OmpA(N21) (with IPTG, but no antibody
staining).*

![FACS result - Sample OmpA(N21) signal](https://static.igem.wiki/teams/5403/wiki/results/results-facs/1a/1a-s-stained-only-signal.png)

**Figure x:** *FACS result of OmpA(N21) (with IPTG and only secondary
antibody incubation).*

shows the signal of the OmpA(N21) sample without IPTG and with
no antibody staining. It is expected that the signal will not
be visible in Q1 and Q2, which is confirmed by the FACS
result.
shows the signal of the OmpA(N21) sample with IPTG, but
without antibody staining. The signal will therefore only come
from the protein-bound GFP. As can be seen in the Figure,
around 99% of the signal is visible in Q4 and Q3. This
suggests that the GFP itself is not sufficient enough for FACS
analysis and the antibody staining is necessary.
is the signal of the OmpA(N21) sample with IPTG and only
secondary antibody. As only around 1.5% of the signal is
present in Q1 and Q2, it can be concluded that no staining
occured. Therefore, it can be concluded that little to no
non-specific secondary antibody staining occured.

![FACS result - Sample OmpA(N21) signal](https://static.igem.wiki/teams/5403/wiki/results/results-facs/1a/1a-p-s-signal.png)

**Figure X:** *FACS result of OmpA(N21) (with IPTG and antibody
staining).*

![FACS result - Sample OmpA(N21) signal](https://static.igem.wiki/teams/5403/wiki/results/results-facs/1a/1a-nc-p-s-signal.png)

**Figure X:** *FACS result of OmpA(N21) (without IPTG, but with
antibody staining).*

For the OmpA(N21) sample with IPTG and antibody staining,
around 9% of the signal is within the color spectrum of the
dye (Q1) in
. This is larger than the sample without antibody staining.

When looking at
, around 4% of the signal is present within the color spectrum
of the dye. This is not expected, as no IPTG is added to this
sample, meaning that the protein of interest should not be
expressed. As the negative control has proven that
non-specific staining does not occur, another explanation must
be found. One could be leaky expression.

Leaky expression in protein expression refers to the
unintended, low-level production of a protein even when the
gene or promotor that controls its expression is supposed to
be repressed or inactive
. In other words, the regulatory system designed to tightly
control protein expression is not fully effective, allowing
for some leakage of expression in the absence of the intended
inducer or under non-inducing conditions. The fact that the
signal is present at a lower quantity than in the IPTG induced
sampe confirms this hypothesis.

There is no clear explanation for why the signal intensity in
the non-induced sample is higher compared to the induced
sample.

##### Conclusion:

Although some leaky expression occurs in the non-induced
sample, a clear staining signal can be seen in the induced
OmpA(N21) sample. As non-specific staining is ruled out, it
can be concluded that the fusion protein consisting of
OmpA(N21) bound to GFP is correctly expressed on the membrane
of *E. coli* BL21 cells.

### FACS - OmpA + GFP

The OmpA membrane protein, fused to GFP, is expressed in BL21
cells by induction of IPTG. The sample is stained with a secondary
antibody conjugated to a dye, to increase the fluorescent signal
of the GFP. The fluorescent signal is analysed with FACS.

##### Aim:

Analyze the presence of GFP, bound to membrane protein OmpA(N21),
on *E. coli* BL21 cells.

##### Methods:

The sample of OmpA bound to GFP is prepared and analyzed in the
same way as the OmpA(N21) sample: antibody staining and analysis
with FACS. The protocol can be found
[here](experiments#FACSprotocol).

##### Results:

The preproccesing of the FACS results is done in the same way as for
the OmpA(N21) sample. First, the results are gated correctly. After
this, doublet discrimination is introduced. Lastly, the placement of
the quartile lines are determined.

![FACS result - Sample OmpA signal](https://static.igem.wiki/teams/5403/wiki/results/results-facs/1b/1b-nc-unstained-signal.png)

**Figure X:** *FACS result of OmpA (no IPTG and no antibody staining).*

![FACS result - Sample OmpA signal](https://static.igem.wiki/teams/5403/wiki/results/results-facs/1b/1b-ustained-signal.png)

**Figure x:** *FACS result of OmpA (with IPTG and no antibody
staining).*

![FACS result - Sample OmpA signal](https://static.igem.wiki/teams/5403/wiki/results/results-facs/1b/1b-s-stained-only-signal.png)

**Figure x:** *FACS result of OmpA (with IPTG and only secondary antibody
incubation).*

shows that, with IPTG induction, the GFP signal itself is not
strong enough for FACS analysis. This is a result that would
also be expected for the sample without IPTG induction, but the
pattern of this sample in
is different from the sample with IPTG. An explanation for this
could be that IPTG induction results in misfolding or
degradation of OmpA. The expression of the OmpA could also
potentially lead to toxicity in the cell, preventing GFP
expression from being detectable.

shows the negative control sample for non-specific binding of
the secondary antibody. When non-specific staining does not
occur, no signal is expected in Q1. But it is visible that 14%
of the total signal is in Q1, suggesting a Cyanine3 staining.
This suggests that the secondary antibody not only binds to the
primary antibody, but also somewhere else on the BL21 cell
membrane.

![FACS result - Sample OmpA signal](https://static.igem.wiki/teams/5403/wiki/results/results-facs/1b/1b-p-s-signal.png)

**Figure X:** *FACS result of OmpA (with IPTG and antibody staining).*

![FACS result - Sample OmpA signal](https://static.igem.wiki/teams/5403/wiki/results/results-facs/1b/1b-nc-p-s-signal.png)

**Figure X:** *FACS result of OmpA (without IPTG, but with antibody
staining).*

When looking at
, around 27% of the sample shows antibody staining. The
intensity of this signal closely resembles that of the secondary
antibody control group. It is therefore difficult to conclude if
this is staining from the antibody complex binding to GFP or
non-specific binding of the secondary antibody.

, the sample without IPTG, shows a different signal pattern.
Although the total amount of signal is lower than in
, the signal intensity is higher. The presence of signal could
be due to leaky expression, but there is no clear explanation
for why the signal intensity is much higher compared to the
induced sample.

##### Conclusion:

The antibody staining of OmpA + GFP shows non-specific secondary
antibody binding. This non-specific staining makes it difficult to
confirm the presence of OmpA + GFP on the membrane of the BL21
cells, especially because the signal of the induced sample shows a
similar pattern as the secondary antibody negative control. On top
of that, the non-induced sample shows a higher intensity than the
induced sample, which cannot be explained by leaky expression
alone.

Due to these uncertainties, it is adviced to redo the exact
experiment to check if the result is the same. If this results in
the same FACS data, a different combination of primary and
secondary antibody can be chosen to hopefully reduce non-specific
secondary antibody binding.

Next to the FACS experiment, it is recommended to perform other
experimental set-ups to identify the presence of the OmpA fusion
protein. These could include visualization techniques such as
super-resolution microscopy (SRM) and immunofluorescence
microscopy (IF), or alternative labelling method like cell surface
biotinylation.

Next to that, the induction system itself can be tested by using
qPCR for mRNA levels or Western blot for protein levels.

### Cloning for *M. smegmatis* proteins

We attempted to linearize the pCHERRY3 plasmid for fusion proteins
(3a) up to (3h) for *M. smegmatis* . The first strategy
included a linearization followed with a Gibson assembly. When
this kept failing, we altered our strategy to a
restriction-ligation.

#### Gibson Assembly

##### Aim:

Linearization of fusion proteins (3a) up to (3h) into the pCHERRY3
plasmid.

##### Methods:

Vector linearization was achieved through PCR, followed by DpnI
digestion and a PCR purification step. The concentration was then
determined using NanoDrop. Subsequently, Gibson Assembly was
performed, and the resulting samples were visualized on an agarose
gel to check whether the correct plasmid was assembled.

##### Results:

![agarose plasmid digestion](https://static.igem.wiki/teams/5403/wiki/results/agarose-gel-psti-hind3-bamhi.png)

**Figure x:** *Agarose gel analysis of pCherry3 plasmid after restriction
enzyme digestion with BamHI, PstI, and HindIII.*

![agarose gel failed pcher3](https://static.igem.wiki/teams/5403/wiki/results/mislukte-gel1.png)

**Figure x:** *Agarose gel showing a failed attempt at linearizing
pCHERRY3*

One step to ease the linearization of a plasmid is to first
digest the plasmid with restriction enzymes. In the case of
pCHERRY3, BamHI, PstI and HindIII were used as seen in
.
show a linearization attempt that was deemed unsuccesful, since
the band of the plasmid is ~500bp instead of the wanted ~6000bp.

##### Conclusion:

We were unable to get the correct plasmid through Gibson assembly,
even with the correct digested pCHERRY3. A more in depth
description about what we tried can be read on our
[engineering page](engineering) and
[notebook page](notebook). Therefore
the strategy was changed from Gibson assembly to
restriction-ligation cloning.

#### Restriction-ligation

##### Aim:

To insert the fusion protein (3c) PorinN50-GFP and the protein
(3h) GFP into pCHERRY3 for expression

#####

Methods:

First, a plasmid digestion was performed on pCHERRY3 with the
restriction enzymes BamHI and HindIII, as seen in
. The restriction of the gBlocks was performed only on fusion
proteins (3c) and (3h) due to limited materials and the
unavailability of the correct primers needed to amplify gBlocks
via PCR. Secondly, a ligation was performed on the digested
pCHERRY3, for fusion protein 3c and protein 3h. We then put the
sample on gel to evaluate whether it succeeded and sent the sample
to Azenta for sequencing.

##### Results:

![agarose gel 3c](https://static.igem.wiki/teams/5403/wiki/results/gel-restriction-ligation-pcher3-1.png)

**Figure x:** *Agarose gel analysis of constructed pCHERRY3 plasmid after
restriction ligation with fusion proteins (3c) porinN50-GFP
and protein (3h) GFP in the pCHERRY3 vector.*

##### Conclusion

The gel showed promising results, indicating that
restriction-ligation succeeded. As seen in
 the biggest band
was around ~6000bp, which could indicate our constructed plasmid.
However, the agarose gel showed contamination in the sample as well.
The Azenta results unfortunately confirmed that the sequence was not
correct. We plan to assemble the plasmids for the expression of the
fusion proteins in *M. smegmatis* in the future (recombinant
plasmids 3a-3g), by restriction-ligation cloning, but with a higher
insert concentration to improve the efficiency of the process. This
way, we might be able to successfully create our recombinant
plasmids and can use them for expression in *M. smegmatis.*

# Post-Insertion

### Background

Two strategies for the functionalization of the bacterial membrane
vesicles were investigated in the laboratory. The second strategy
to be discussed is the post-insertion strategy.

![PostInsertion](https://static.igem.wiki/teams/5403/wiki/results/results-postinsertion/postinsertion.png)

**Figure 1:** *Post-insertion method: BMVs are isolated using
ultracentrifugation. Micelles are functionalized with a
Spycatcher using Click Chemistry. The
Spycatcher-functionalized micelles and the BMVs are then
mixed, allowing the Spycatcher to insert into the BMV
membrane. The BMV is functionalized by adding the antigen
that is fused to the Spytag.*

The post-insertion method, in contrast to the genetic engineering
method, requires isolation of BMVs before functionalization. After
isolation, BMVs will be combined with micelles functionalized with
Spycatcher proteins, allowing the Spycatcher to integrate in the
membrane of the BMV. Unlike the genetic engineering approach—where
GFP was fused as a model antigen before introducing modularity—in
this method, Spycatcher is directly incorporated into the BMVs.
This Spycatcher functionalization of our BMV establishes a
‘plug-and-play’ system for antigen integration. It enables the
development of off-the-shelf, personalized vaccines, particularly
for cancer treatment, where the desired antigens can be easily
‘clicked’ onto the BMVs.

In order to assemble micelles with Spycatcher on the surface, the
amino acid sequence of Spycatcher was adjusted such that a unique
cysteine residue exists at its N-terminus ((2a) CysSpycatcher).
The cysteine is used to attach the phospholipid DSPE-MAL by
maleimide coupling (Click Chemistry). The Spytag is fused to GFP
to verify functionalization. An overview of the protein inserts,
plasmids and hosts for obtaining the proteins necessary for this
method can be found in Table 2.

**Table 2:**
*Overview of proteins used for the post-insertion method. The
table lists the different protein inserts cloned into various
plasmid backbones, their corresponding host organisms.*

| ID | Protein inserts | ID (parts registry) | Plasmid | Host |
| --- | --- | --- | --- | --- |
| 2a | CysSpycatcher + 6H | BBa\_K5403005 | pET28a | E. coli |
| 2b | Spytag + GFP + 6H | BBa\_K5403021 | pET28a | E. coli |

### Protein expression and purification in *E. coli*

We purified CysSpycatcher and Spytag-GFP proteins for use in the
post-insertion method, which will allow functionalization of
micelles or bacterial membrane vesicles with Spycatcher for
subsequent antigen binding through Spytag.

##### Aim:

Express and purify proteins that will be employed for the
post-insertion method.

##### Methods:

The cloning of the plasmids for the expression of CysSpycatcher
and GFP-Spytag was performed in a pET28a vector by Gibson
assembly. The assembled plasmids were multiplied in
*E. coli* TOP10 cells and miniprepped. CysSpycatcher and
Spytag-GFP were expressed in BL21 cells, followed by
IMAC-purification. The resulting samples were visualized on an
SDS-PAGE gel.

##### Results:

![SDS page gel](https://static.igem.wiki/teams/5403/wiki/results/sds-page-gel-protein-purification.png)

**Figure X:** *CysSpycatcher and Spytag-GFP proteins are expressed in E.
coli and purification with Ni2+-affinity chromatography
afforded pure proteins, as shown by SDS-PAGE analysis. L:
molecular weight ladder, Lys: cell lysate, FT: flow
through, W1: wash fraction 1, W2: wash fraction 2, E1:
elution fraction 1, E2: elution fraction 2*

##### Conclusion:

It can be seen from the SDS-PAGE gel that the purification was
successful, resulting in a band at 9.9 kDa for CysSpycatcher and
a band at 29.6 kDa for Spytag-GFP.

### CysSpycatcher coupling with Spytag-GFP

We coupled Spytag-GFP and CysSpycatcher and ran them on an
SDS-PAGE gel to verify the successful conjugation of the two
proteins, ensuring that the functional domains of Spytag and
Spycatcher interact correctly, forming a stable covalent bond.

##### Aim:

Check if the recombinantly engineered CysSpycatcher and Spytag-GFP
are able to form a covalent isopeptide bond.

##### Methods:

To couple CysSpycatcher with Spytag-GFP, we prepared the proteins
in a 1:1 molar ratio. The two components were mixed in a reaction
buffer (e.g., PBS or Tris buffer, pH 7.4) and incubated at room
temperature to allow the covalent bond formation between
Spycatcher and Spytag.

##### Results:

The SDS-PAGE analysis in
reveals several key observations. In the lanes containing
CysSpycatcher and Spytag-GFP, the expected bands for each
individual protein are present. CysSpycatcher appears as a band
around 10 kDa, while Spytag-GFP shows up at approximately 30 kDa.
In the lanes where the two proteins were mixed, a prominent new
band appears around 40 kDa, consistent with the expected molecular
weight of the CysSpycatcher-Spytag-GFP conjugate.

However, there is an unexpected excess of unbound Spytag-GFP in
the conjugate lanes, with almost no detectable free CysSpycatcher
remaining, despite mixing in a 1:1 molar ratio. This suggests that
the CysSpycatcher is fully utilized in binding Spytag-GFP, but for
some reason, the Spytag-GFP is in excess. One possible explanation
for this observation is inaccuracies in protein quantification or
loss of CysSpycatcher during sample preparation.

Additionally, a band is observed around 75 kDa, which could
represent one CysSpycatcher protein coupled to two Spytag-GFP
proteins. This may indicate that CysSpycatcher is binding more
than one Spytag-GFP molecule, possibly due to an incorrect molar
ratio during mixing or due to multimerization of proteins. This
suggests that optimizing the ratio and reaction conditions could
improve the specificity and efficiency of the coupling, preventing
the formation of higher molecular weight complexes.

![SDS page gel](https://static.igem.wiki/teams/5403/wiki/results/spycatcher-spytag.png)

**Figure X:** *SDS-PAGE gel showing successful covalent coupling between
CysSpycatcher (~10 kDa) and Spytag-GFP (~30 kDa). The
conjugated product (~40 kDa) is visible in the lanes
containing the mixture after incubation, confirming the
formation of the CysSpycatcher-Spytag-GFP complex.*

##### Conclusion:

Although the coupling reaction and conditions need further
optimization, our results demonstrate that CysSpycatcher and
Spytag-GFP successfully combine, as evidenced by the appearance
of the expected conjugate band on the SDS-PAGE gel. The presence
of higher molecular weight complexes and excess Spytag-GFP
suggests room for improvement in the reaction setup, but the
fundamental Spycatcher-Spytag interaction has been clearly
shown.

### Micelle formation

We explored the use of DSPE-Mal to form micelles and tested
whether CysSpycatcher could be successfully attached. We tried to
confirm this attachment using Spytag-GFP. While the results showed
some signs of successful functionalization, both the micelle
formation and protein attachment were inconsistent, highlighting
the need for optimization in the protocol to achieve more reliable
and stable micelle formation and functionalization.

##### Aim:

Make micelles that are functionalized with Spycatcher as
preparation for the post-insertion method.

##### Methods:

We prepared micelles using only DSPE-Mal due to the unavailability
of DSPE, deviating from the typical DSPE ratio of 4:1. To form the
micelles, DSPE-Mal was dissolved in methylene chloride, and a
lipid film was created by evaporating the solvent under a nitrogen
stream. The lipid film was then hydrated with an appropriate
buffer and sonicated to produce micelles.

For functionalization, CysSpycatcher was added to the micellar
solution and incubated, allowing the CysSpycatcher to covalently
attach to the DSPE-Mal micelles. The micellar solution was then
filtered using a 50 kDa centrifugal filter to remove any unbound
CysSpycatcher. Next, the filtered DSPE-Mal-CysSpycatcher micelles
were incubated with Spytag-GFP to facilitate covalent binding
between Spycatcher and Spytag. This solution was filtered again
with a 50 kDa centrifugal filter to eliminate any unbound
Spytag-GFP. To verify successful functionalization, the
DSPE-Mal-CysSpycatcher + Spytag-GFP micelles were compared with a
negative control, where DSPE-Mal micelles were directly incubated
with Spytag-GFP and filtered using a 50 kDa centrifugal filter to
remove unbound Spytag-GFP.

The structural properties of the DSPE-Mal micelles and the
unfiltered/filtered DSPE-Mal-CysSpycatcher micelles were evaluated
using DLS to assess their size distribution. Additionally, the
filtered solution containing DSPE-Mal-CysSpycatcher + Spytag-GFP
was compared to the DSPE-Mal + Spytag-GFP negative control using
fluorescence analysis to confirm successful functionalization and
binding of Spytag-GFP to the CysSpycatcher-modified micelles.

##### Results:

Table 3 shows the PdI (Polydispersity Index) and Number Mean
(d.nm) for the DSPE-mal micelles and DSPE-mal-CysSpycatcher
micelles, both filtered and unfiltered.

**Table 3:**
*DLS results of the DSPE-mal micellar solution and the
DSPE-mal-CysSpycatcher micellar solution after filtering.*

| Sample Name | PdI | Number Mean (d.nm) |
| micelles DSPE-mal 1 | 0.4 | 167.1 |
| micelles DSPE-mal 2 | 0.434 | 45.29 |
| micelles DSPE-mal 3 | 0.592 | 51.66 |
| micelles DSPE-mal + CysSpycatcher unfiltered 1 | 0.367 | 42.12 |
| micelles DSPE-mal + CysSpycatcher unfiltered 2 | 0.401 | 58.95 |
| micelles DSPE-mal + CysSpycatcher unfiltered 3 | 0.426 | 60.85 |
| micelles DSPE-mal + CysSpycatcher filtered 1 | 0.262 | 18.68 |
| micelles DSPE-mal + CysSpycatcher filtered 2 | 0.254 | 62.38 |
| micelles DSPE-mal + CysSpycatcher filtered 3 | 0.269 | 25.6 |

The DLS data reveal significant variability in the size
distribution and polydispersity of both the DSPE-mal micelles and
the DSPE-mal-CysSpycatcher micelles, with PdI values ranging from
0.4 to 0.592 for DSPE-mal micelles and 0.254 to 0.426 for
DSPE-mal-CysSpycatcher micelles, indicating that all the solutions
are highly heterogeneous. While filtration somewhat improves
homogeneity, as evidenced by a reduction in PdI values, the number
mean size still varies greatly across the samples (e.g., from
18.68 nm to 167.1 nm), suggesting inconsistent micelle formation.
This inconsistency highlights the need for the inclusion of DSPE
in the formulation, as it plays a crucial role in stabilizing the
micelles. Additionally, the protocol for micelle formation
requires further optimization to produce uniform and stable
particles.

Fluorescence measurements were taken to assess whether Spytag-GFP
successfully bound to the DSPE-mal micelles functionalized with
CysSpycatcher. The fluorescence signal was measured for three
samples: a blank (3 RFU), DSPE-mal-CysSpycatcher + Spytag-GFP
(50,465 RFU), and DSPE-mal + Spytag-GFP (27,103 RFU).

The significantly higher fluorescence signal from the
DSPE-mal-CysSpycatcher + Spytag-GFP sample (50,465 RFU) compared
to the DSPE-mal + Spytag-GFP (27,103 RFU) and the blank (3 RFU)
suggests that the micelles were successfully functionalized with
CysSpycatcher, allowing the covalent binding of Spytag-GFP. This
increase in fluorescence likely indicates the successful
attachment of Spytag-GFP to the CysSpycatcher-functionalized
micelles.

However, it is unusual that a substantial fluorescence signal
remains in the DSPE-mal + Spytag-GFP sample, even after filtering
with a 50 kDa centrifugal filter. Since Spytag-GFP is a small
protein (~30 kDa), it should have been removed during filtration
if it were unbound. This persistent fluorescence suggests that
Spytag-GFP might be adhering to the micelles non-specifically,
even in the absence of CysSpyCatcher. This could be due to
hydrophobic interactions or other surface characteristics of the
micelles that cause the Spytag-GFP to stick, rather than being
fully removed by filtration.

##### Conclusion:

The DLS and fluorescence data suggest potential for forming
DSPE-mal micelles functionalized with Spycatcher, but micelle
formation was inconsistent and showed high variability in size
and polydispersity. Non-specific binding of Spytag-GFP was also
observed. Overall, this exploratory protocol holds promise but
requires significant optimization at each step—micelle
formation, functionalization, and filtration—to achieve
consistent and reliable results for future applications.

## References

Images created with BioRender.com

{% endblock %}