E. coli Nissle 1917

Investigating a suitable chassis for biofilm formation

Thurday 07/07/11

Nissle 1917 cells were extracted from the Mutaflor tablets, which were obtained from Gerhard Breves, of Hanover, Germany.

A single Mutaflor tablet was dissolved in 10ml of distilled water and serial dilutions were made of this solution. 200µl of each dilution were plated on MacConkeys agar and incubated at 37ºC overnight. A negative control of undiluted extract was plated on LB.

The approximate predicted cell count of the dilutions is as follows:

Undiluted 25 x 10⁹ cells /10ml10⁻1 x dilution 25 x 10⁸ cells /10ml10⁻! x dilution 25 x 10⁷ cells /10ml10⁻" x dilution 25 x 10⁶ cells /10ml10⁻⁴ x dilution 25 x 10⁵ cells /10ml10⁻⁵ x dilution 25 x 10⁴ cells /10ml10⁻⁶ x dilution 25 x 10" cells /10ml10⁻⁷ x dilution 25 x 10! cells /10ml10⁻⁸ x dilution 250 cells /10ml10⁻⁹ x dilution 25 cells /10ml

(Values obtained from the information leaflet inside the Mutaflor tablet packet)

Serial Dilutions: All serial dilutions are performed by preparing a relevant number of microfuge tubes containing 900µl of relevant media. 1000µl of undiluted culture is added to a fresh microfuge tube, this is then vortexed for a few seconds and 100µl is inoculated into the next microfuge tube. Repeat this for as many dilutions as are required using a fresh tip for each inoculation.

Friday 08/07/11

Colony counting of the serial dilutions yielded the following results:

Undiluted LB NG (No Growth – no colonies were observed)Undiluted Mc LG (Lawn Growth - density of colonies too high to count)10⁻1 x dilution LG10⁻! x dilution LG10⁻" x dilution 115 cells10⁻⁴ x dilution 22 cells10⁻⁵ x dilution 6 cells10⁻⁶ x dilution NG10⁻⁷ x dilution NG10⁻⁸ x dilution NG10⁻⁹ x dilution NG

No growth was seen on the plain LB plate, as a result it was assumed that Nissle cells do not grow on LB.

The inaccurate values obtained for serial dilutions were attributed to cell 'clumping', this issue was also encountered in the biofilm assay and was tackled by low power sonication of the sample for 30 seconds before plating.

Addition: It was later found that Nissle cells readily grow on LB. The above false negative is attributed to the use of a mislabelled LB ampicillin plate

Monday 11/07/11

MacConkeys broth overnight cultures were set up from the Nissle tablet extraction.

Overnight Culture: All overnight cultures are set up as a single picked colony inoculated into a corning tube containing 5ml of media and any relevant antibiotics.

Tuesday 12/07/11

Using the overnight cultures, the first biofilm assay using the Nissle was set up, measuring biofilm growth over a period of 24 hours. Full information on the assay used is available in the protocols section of the website. Please note that MacConkey broth was used in place of LB for this protocol.

Four glycerol stocks of Nissle E.coli were prepared.

Glycerol Stocks: These are prepared by mixing 750µl of over-night culture with 250µl of 60% glycerol. The samples are then transferred to the -80 freezer for storage.

Thursday 14/07/11

Nissle cells returned positive results for biofilm formation. Following this positive result, overnight cultures were set up for the creation of chemically competent Nissle cells

Friday 15/07/11

10ml of overnight culture was made competent. Full information on the protocol used is available on the protocols section of the website. Please note that MacConkey’s broth was used instead of LB for this protocol.

These competent cells were then tested by transforming with a previously used RFP construct at 10-fold dilution, as well as the crtb6 plasmid as positive control. The transformation protocol used is available on the protocols section of the website. Please note that MacConkey’s broth was used instead of LB for this protocol.

The plates from these transformations were left to grow at room temperature on the bench, due to limited access to the lab over the weekend.

Monday 18/07/11

The weekend transformation yielded no positive results. 200ml of untransformed competent cells were plated MacConkey agar to test viability.

The transformation plates from the weekend were incubated at 37 degrees to see if any further growth would become apparent.

Tuesday 19/07/11

Following incubation, the Nissle transformations still showed no growth. The untransformed chemically competent cells showed proliferative growth on MacConkey, indicating that chemical competence induction had failed.

Three overnight cultures of Nissle were set up, 2 intended for making electrocompetent cells and 1 for a biofilm assay. An overnight of P. aeruginosa was also set up for biofilm comparison.

Wednesday 20/07/11

The overnight cultures were made electrocompetent This generated 2ml of electrocompetent cells, which were divided into aliquots of 50µl and stored at -80ºC.Full information on the protocol used is available on the protocols section of the website. Please note that MacConkey broth was used instead of LB for this protocol.

Thursday 28/07/11

The electrocompetent Nissle cells were transformed with RFP and puC19 as positive control via electroporation. Full information on the protocol used is available on the protocols section of the website. Please note that MacConkey broth was used instead of LB for this protocol.

Each transformation was carried out at serial dilutions ranging from undiluted to 10-2,. Three samples arced.

The kit- extracted registry RFP DNA had dried up. It was resuspended with water and re-used.After incubation, 200µl of transformed cells were plated on ampicillin MacConkey plates, the remaining 800µl of transformants were refrigerated overnight.

Friday 29/07/11

Positive growth was observed on all puC19 plates which did not arc, no growth was seen of any RFP transformants. The remaining 800µl of transformation was plated and left on the bench over the weekend.puC19 positive control transformants were streaked out on:- LB agar- LB agar + Ap- MacConkeys- MacConkeys +ApNissle was expected to grow only on MacConkeys, and MacConkey’s + Ap.

Monday 01/08/11

The positive control that was streaked on plates yesterday grew just as happily on LB as on MacConkeys. It was decided that rigorous testing would be carried out to search for resistances naturally present in Nissle E.coli.

Nissle cells were extracted from a fresh Tablet, and were again plated in serial dilution, this time on both, MacConkey’s and LB (100µl of each dilution). Dilutions ranged from undiluted to 10⁻⁶.

MacConkey’s MacConkey’s + Amp

LB Agar LB + Amp LB + Cm

Nissle E.comp ✓ ! ! ! !

Nissle Chem.comp ✓ ! ! ! !

Nissle Tablet extraction (10⁻!) ✓ ! ! ! !

Nissle puC19 transformant ✓ ✓ ! ! !

P.putida (positive control) ✓ ✓ ✓ ✓ ?

Water ! ! ! ! !

Blank ! ! ! ! !

Table 1: Expected results

To plate the puC19 positive control 1 colony of growth was scooped into 350µl of water for plating later.We also set up overnight cultures of the positive control as we were planning to digest and run a gel.

Tuesday 02/08/11

We performed a miniprep on the overnight culture of the positive control. Interestingly, Nissle that have been grown in MacConkey’s change colour during the miniprep.(pictures of funky colour changes)

MacConkey’s MacConkey’s + Amp

LB Agar LB + Amp LB + Cm

Nissle E.comp ✓lawn

! ✓lawn growth

! !

Nissle Chem.Comp ✓5 colonies

! ✓61 colonies

! !

Nissle Tablet extraction (10⁻!)

! ! ✓122 colonies

! !

Nissle puC19 transformant

✓lawn

✓lawn

✓lawn growth

✓lawn growth

!

P. putida !15 colonies

! ✓near-lawn

✓979 colonies

!

Water ! ! !2

(contamination)

! !

Blank !1 (fuzzy)

! !6

(contamination)

! !

Table 2: Observed results

As the RFP did not grow at all we suspect that this is due to the DNA being dried up before.We repeated the electroporation usingpUC19 (undiluted, 10-fold, 100-fold, 1000-fold dilution)RFP construct from miniprep (undiluted, 10-fold, 100-fold, 1000-fold dilution)plus a negative control.We performed the electroporation according to protocol, using MacConkey’s thoughout, and plated both 200 and 800µl on the transformations onto MacConkey’s + Amp.

We set up overnight cultures of the plain Nissle cells from the tablet extraction, three each in LB broth and MacConkey’s broth.

Wednesday 03/08/11

We got growth for the electro-tranformations of Nissle with the RFP! However, the colonies look a bit funny, grew in big clumps and frequently showed two colours (yellow and purple, the yellow being a degradation product of amino acid metabolism), and were therefore restreaked onto more antibiotic plates to check resistance, and restreaked to try and get single colonies. Nissle cells have no innate resistance to antibiotics.

puC19 RFP

200μl 800μl 200μl 800μl

Undiluted clumped growth

clumped growth

arced arced

x 10⁻¹ x x lawn, yellow on the inside, with purple edges

x

x10⁻² x x lawn, yellow on the inside, with purple edges

Red lawn, unevenly spread

x 10⁻³ x x lawn, yellow on the inside, with purple edges

one large purple patch surrounded by clumped colonies

Table 3: description of growth of Nissle transformants from 03/08/11

Clumping of cells is believed to be due to Nissle cells being very adept at clinging to each other. A quality that also allows them to form biofilms. We recommend sonicating or throughly vortexing the cells before plating them.

We examined the RFP transformants under the Mircoscope with gram stain, and compared them to pUC19 transformants under the fluorescence microscope. Both results were confusing and need to be checked again.

We restreaked a selection of RFP and puC19 transformants onto LB plain, LB ampicillin, LB chloramphenicol, LB kanamycin, MacConkeys plain, MacConkeys ampicillin and MacConkeys kanamycin.

We retried making chemically competent Nissle cells because Andrew does not believe in the ones we already have. The protocol was altered because we were using a different centrifuge to spinning a 20ml culture down at 4000rpm for 5 minutes.

There was also a slight delay in getting these cells into the -80ºC freezer.

We made glycerol stocks of Nissle Tablet extraction overnights set up yesterday.We also set up a 24h biofilm assay using the same overnights as for the glycerols.

We set up overnight cultures of what we think are the RFP transformants in Carbenicillin MacConkey’s. The overnights were inoculated with single purple colonies and yellow lawn (not single colonies, we’ll go to biologist-hell for that)

For the overnight cultures we picked:- RFP x 10⁻# 200µl purple- RFP x 10⁻# 200µl yellow- RFP x 10⁻! 200µl purple- RFP x 10⁻! 200 µl yellow- RFP x 10⁻! 800µl - RFP x 10⁻" 200µpurple- RFP x 10⁻" 200µl yellow- RFP 10⁻" 800µl

We digested yesterday’s miniprep of the positive control and ran it on a gel. The results looked a bit confusing and will be analysed in more detail tomorrow.

Thursday 04/08/11Only the streaks that originated from the 800µl plating showed growth for RFP in the streaking for single colonies. The 200µl RFP streak showed no growth, while the pUC19 control grew fine, however, there was clumping here too. We should sonicate the cells before plating them.

Contradictory, there was no growth on the resistance streak for any of the RFP transformants.However, in the overnights that were set up yesterday it was again the cultures from the 800µl plates that grew, while all other RFPs showed no growth. Glycerol stocks were set up of the overnights that showed growth. The same overnights were then miniprepped and digested with XbaI and SpeI, along with one of the old minipreps of RFP for comparison. No DNA was visible on the gel.We set up overnights of RFP again, this time in MacConkey’s broth and LB broth with carbenicillin.

We tried the new chemically competent cells we made using pUC19, RFP (undiluted, 10-fold, 100-fold, 1000-fold dilution) and GFP (undiluted, 10-fold, 100-fold, 1000-fold dilution)

The resulting 1ml culture of transformants was plated half-and-half on LB and MacConkey’s with Carbenicillin.

Friday 05/08/11

Fittingly for Ellin’s Birthday we found growth for the GFP transformants from yesterday. Also, some of the overnights that were set up yesterday showed definite expression of RFP, especially the ones set up in LB, like the one shown . Glycerol stocks were made of these overnights.

30µl of the RFP-expressing overnights was tested for fluorescence under the microscope and compared to pUC19 transformants from Wednesday. Both of them showed definite red fluorescence, while no flurescence was visible for pUC19. An overnight of pUC19 is to be set up on Monday, to check for fluorescence again.

We performed minipreps of the RFP overnights and stored them in the fridge.

Monday 08/08/11

Loaded a gel with digests of our minipreps from Friday (digested with XbaI and SpeI). Also ran some of Pietro’s PCR product. However, the gel that was used was too old, and the SybrSafe had gone off. Fortunately Andrew though of staining the gel with Ethidium Bromide, which allowed us to visualise the bands after all, showing us that they were in fact RFP.We set up overnight cultures of GFP Nissle, RFP Nissle and mixed GFP and RFP Nissle for biofilm formation.

Tuesday 09/08/11

We were very much inconvenienced by a power cut and only managed to prepare minipreps of GFP and to set up a biofilm assay (the usual protocol) of GFP and RFP Nissle. and leave on desktop shaker over night.Glycerols were also made of the GFP overnights.

Figure 1: Overnight culture of E.coli Nissle 1917 transformed with and expressing red fluorescent protein.

Thursday 11/08/11

We examined the GFP biofilms. The colours are unusual. We are certain that MacConkey’s Broth will distort fluorescence, and will make red fluorescence look orange, while green fluorescence looks yellow. This can be very misleading, therefore CAUTION!To have a better look at this fluorescence we set up new overnights of Nissle GFP (form 04/08) and Nissle RFP (from 02/08) in LB + Carbenicillin.We used:RFP 100x and 1000x dilution 800µl plates from 02/08GFP Undiluted plate colonies 4 and 10 from 04/08

Friday 12/08/11

We could not get the fluorescence microscopes to work, and assumed that the light bulbs had given up. Examining fluorescing Nissle cells was therefore discarded.

Wednesday 31/08/11

We set up overnight cultures of other transformed and non-transformed E.coli strains ie Top10, BL21 and Top10 transformed with I15009, in MacConkeys to check if the colour changes observed when miniprepping Nissle are due to the cells, or due to the MacConkey medium.

Thursday 01/09/11

When miniprepping yesterdays overnights the same colour changes as in Nissle were observed, though they were not nearly as vibrant as with Nissle.

Thursday 08/09/11

We continued investigation on the Biofilm forming properties of the Nissle strain by setting up 16 hour and 24 hour biofilm in the usual set up. We intend to use these to quantify the biofilms - like Chris did with Pseudomonas -, to stain them, and to get some pictures through the fluorescent microscope and the electron microscope. We use a restreak of our RFP transformants from 03/08 to set up the biofilms, as well as puC19 transformants and older glycerol stocks of plain Nissle as controls. The plates are shown in figure 2, with clearly visible RFP expression on plate 1.

Friday 09/09/11

We scraped the biofilms off the slide and vortexed them for 1 minute each, as we no longer have access to a working sonicator. Ellin then diluted the resulting culture, as well as the culture of cells that disscoiated from the biofilm in serial dilutions ranging from undiluted to x 10⁻⁸. The plates were left in the incubator until Saturday, and then moved into the fridge.

Monday 12/09/11

The colonies on the plates were counted to obtain an estimate of the number of cells per ml of culture.

16 hour biofilm

Dispersal Biofilm1 2 3 1 2 3

undiluted lawn lawn lawn lawn lawn lawn

x 10⁻" lawn lawn lawn lawn lawn lawn

x 10⁻# 2291 2894 4547 3911 5278 3657

x 10⁻! 536 536 401 378 352 618

x 10⁻⁴ 70 70 42 67 69 75

x 10⁻⁵ 5 4 8 8 12 11

x 10⁻⁶ 2 3 0 0 0 0

x 10⁻⁷ 0 0 0 0 0 0

x 10⁻⁸ 0 0 0 0 0 0Table 3: Colonies of 16 hour dispersal and biofilm colonies in serial dilution

24 hour Biofilm

Dispersal Biofilm1 2 3 1 2 3

undiluted lawn lawn lawn lawn lawn lawnx 10⁻" lawn 5438 lawn lawn 6264 lawnx 10⁻# lawn 929 lawn lawn 750 lawnx 10⁻! lawn 157 lawn lawn 62 lawnx 10⁻⁴ lawn / lawn lawn 13 lawnx 10⁻⁵ lawn 1 9 lawn / 5x 10⁻⁶ lawn / / lawn / /x 10⁻⁷ / / / / / /x 10⁻⁸ / / / / / 0

Table 4: Colonies of 24-hour dispersal and biofilm in serial dilution (dashes indicate clumping of cells, making counting impossible)

In the 24-hour biofilm a lot of clumping occured which made counting next to impossible. We would therefore recommend sonication over vortexing whenever possible. We may increase time spent vortexing to make sure we can properly break up the biofilm before plating.

Today we also set up a 1-hour, 2-hour and 4-hour biofilm, scraped and vortexed as before, and plated in serial dilution ranging from undiluted to x 10⁻".

Tuesday 13/09/11

1-hour Biofilm

Dispersal Biofilm1 2 3 1 2 3

undiluted 3498 2449 2226 2703 2417 2830

x 10⁻" 1177 859 1049 795 1049 350

x10⁻# 45 39 25 40 36 18Table 5: Colony-count for 1-hour dispersal and biofilm growth

2-hour Biofilm

Dispersal Biofilm1 2 3 1 2 3

undiluted 1781 / 3275 3371 / 2957x 10⁻" 795 477 540 636 763 350x10⁻# 29 47 91 20 35 80x 10⁻! 0 2 4 1 16 1

Table 6: Colony-count for 2-hour dispersal and biofilm growth

4-hour Biofilm

Dispersal Biofilm1 2 3 1 2 3

undiluted lawn lawn lawn lawn lawn lawnx 10⁻" 1017 1049 1208 1844 3243 1876x10⁻# 61 190 127 127 413 286x 10⁻! 0 1 10 0 43 33

Table 7: Colony-count for 4-hour dispersal and biofilm growth

Wednesday 14/09/11

We finally got beautiful fluorescing pictures of Nissle biofilms, along with controls. It appears that Nissle cells fluoresce green under certain wavelengths. The use of a confocal microscope may therefore be beneficial.

Figure 3: Comparison of RFP and non-RFP E.coli Nissle biofilms in light and fluorescence microscopy

As can be seen from the above figures 3 and 4, RFP clearly shows up as red fluorescence in the biofilm. Due to what appears to be innate green fluorescence, using green or yellow fluorescent protein may not be practical unless using a confocal microscope. We also set up and fixed more biofilms, for taking pictures with the Scanning Electron Microscope on Monday

Figure 4: Comparison of RFP and puC19 transfromed biofilms in light and fluorescent microscopy

Monday 19/09/11

Term has started. We prepared a graph of our results for the Nissle cell biofilm formation, which shows that planktonic cells and cells within the biofilm multiply at approximately the same rate.

Dispersal Biofilm

0

2250000

4500000

6750000

9000000

1 hour 2 hour 4 hour 16 hour

Cell count Dispersal vs Biofilm

Graph 1 : The increase of cell number within the biofilm and in planktonic cells. Similar increase on cell numbers was recorded for both, showing that biofilm growth increases at a similar rate to planktonic cells.

We also had pictures of multiple 24-hour Nissle biofilms taken by SEM. They clearly show the extracellular matrix that holds the cells together, both when they are further apart (Image 13) or closely packed (Image 14).

Tuesday 20/09/11

Prepare stab cultures of Nissle cells to send off to the registry.

Wednesday 21/09/11

Send off stab cultures from yesterday to the registry for use as a new chassis.

Image 14: Electronmicrograph 10000x of E.coli Nissle 24-hour biofilm, showing the extracellular matrix holding the biofilm closely together.

Image 13: Electronmircograph 5000x of a 24-hour E.coli Nissle biofilm, showing the cells’ long fimbriae.