Team:Brno Czech Republic/Experiments

WET LAB

This page will give you detailed information about the experiments we have performed. You can read about the reasons that led us to design and then execute the experiment, its theoretical background, used materials and methods, and we also give a brief description of our workflow. The protocols we followed are a part of this chapter. All of them can be found in the Protocols section. For each experiment we add a hyperlink to the protocol used.

At Masaryk University, we were allowed to work in a laboratory that is usually used for teaching practical courses during the semester. There, we had at our disposal any equipment we need - flow boxes, cyclers, centrifuges, pipettes and many others. We also had the support of PhD students and technicians from the Experimental biology department, to whom we are very grateful for their advice.

The results of each experiment can be found in the Results section.

Basic experiments

Preparation of growth media

The protocols for media preparation can be found here: PROTOCOL: Preparation of growth media.

Preparation of vectors with our synthetic constructs

1. Preparation of commercial vector with synthetic constructs

Motivation

For the transformation of B. subtilis, we had 4 synthetic constructs prepared by company IDT within the offer mediated by iGEM contest.

These synthetic modules were named A, B, C and D and are further described in the Project design section. Unlike last year, when we worked with linearized construct segments, this year we received synthetic constructs incorporated inside pUCIDT plasmids.

Short description

Synthetic genes were shortly centrifuged in short spin mode and then resuspended in appropriate volume of nuclease free water in a flowbox. Resuspended genes are less stable, so they had to be stored on ice or put in a freezer at -20 °C. We worked with them only inside a flowbox to minimize the risk of DNA contamination. For gene stocks, it is better to use pipette tips with filters as pipettes might contain aerosols from previous pipetting. Any contamination could devalue the whole stock.

2. Preparation of primers

Motivation

Within the PHOSCAGE project we worked with many primers. We mainly used the primers we received last year. Their detailed description can be found here.

Short description

Our primers were also synthesised by the IDT company. They had to be resuspended in nuclease free H2O according to the instructions provided. When working with the primers, we always used a 10 nM solution that was prepared from 100 nM stock solution. The stock solution was made by resuspending the delivered powder. We assigned a number to each primer so that we could easily distinguish them.

Preparation of glycerol stocks and destination vectors for cloning

Protocols are available here (PROTOCOL: Preparation of glycerol stocks ) and here (PROTOCOL: Isolation of plasmids ).

Last year, we received our strain of B. subtilis and the vectors for our project from Dr. Libor Krásný, the head of the Laboratory of Microbial Genetics and Gene Expression at the Academy of Sciences of Czech Republic. For this year's project, we used the same strains of B. subtilis from the glycerol stocks we had prepared a year ago. We also isolated the same destination vectors from last year’s stocks of E.coli. The preparation of these glycerol stocks is mentioned in our wiki from last year.

Plasmid isolation

During our project we often needed to isolate plasmids with the desired vectors from our bacteria. For this purpose, the PureYield™ Plasmid Miniprep System by Promega Corporation was used, where the plasmid DNA was obtained through column purification. For some useful notes or advice visit the protocol Isolation of plasmids.

Restriction digestion

Restriction digestion was needed several times in our wet lab experiments. It was used when our constructs had to be separated from a particular plasmid so that it could be separated and inserted into another vector, and when the length of the restriction fragments needed to be adjusted so that they would be further apart on the gel after electrophoresis. This method was further intended, also, for moving individual constructs between each other or for identifying DNA using typical restriction sites and restriction fragment sizes.

For digestion of plasmids isolated by the method described above, only enzymes with permitted restriction sites were used, namely BamHI, HindIII and BsaI. The table describes use of enzymes and their combination with buffers. Restriction digestion was carried out according to the procedures indicated by the manufacturer.

For some useful notes or advice visit the PROTOCOL: Proof of transformation of Escherichia coli.

Commercial vector with construct	RE	Buffer	Digestion time (min)	Inactivation time (min) (80 °C)
pUCIDT_B	HindIII, BamHI (Thermo Fisher Scientific)	Fast digest buffer	25	15
pUCIDT_C	HindIII, BamHI (Thermo Fisher Scientific)	Fast digest buffer	25	15
pUCIDT_D	HindIII, BamHI, BsaI (NEB)	CutSmart	15	20

Table 1 : Restriction digestion according to the digested vector.

Ligation

T4 ligase was used to link sequences with complementary sticky ends cleaved by restriction enzymes. The ligation is mentioned in PROTOCOL: Preparation of genetic modules in Escherichia coli cloning/expression host.

We had the best experience with overnight ligation.

PCR

Here we describe two modifications of the PCR we used.

Colony PCR was applied only to E. coli (in the case of B. subtilis the E. coli chromosome must first be isolated). GoTaq Master Mix by Promega was used for colony PCR. The temperature and duration of each phase was related to the length of the sequences and primers.

LongPCR was used to verify integration into the chromosome in B. subtilis. Chromosome isolation had to occur first (described above).

When performing Long-Range PCR, we followed a protocol that came with the GoTaq® Long PCR Master Mix by the company PROMEGA here. However after a while we ran out of the Master Mix, so after testing it first we continued using the GoTaq G2 DNA polymerase from PROMEGA. For additional information, visit PROTOCOL: Long-Range PCR. All designed primers we used in PCR reactions are described here.

Purification from gel

Electrophoresis is an effective separation method. The separated DNA needs to be purified from the agarose gel. The protocol used for this was the Wizard® SV Gel and PCR Clean-Up System. To obtain higher concentration of your DNA, you can elute it using a smaller amount of nuclease free water.

Preparation of Escherichia coli cloning host

1. E coli JM109

Testing of competent cells E. coli JM109

Motivation

This experiment was performed in order to test effectiveness of the purchased competent cells. This was accomplished by transforming the empty vectors pDG3661 and pDG1664 into these competent cells.

Short description

Competent cells - E. coli JM109 purchased from Promega company - were used for testing. These competent cells were not prepared by us. They were handled according to this protocol: E. coli Competent Cells Quick Protocol #9FB035, which is available on the website here. For testing competent cells, we used empty vectors pDG1664 and pDG3661. The transformation mixture was carefully plated on LB medium plates containing the corresponding antibiotic - in both cases ampicillin (final conc. 150 µg/ml).
A more specific description of the transformation of these E. coli JM109 cells will be presented in the following subsections.

Transformation of JM109 with pUCIDT plasmids with constructs B, C and D

Motivation

Transformation of E. coli JM109 with our constructs incorporated inside pUCIDT plasmids with the aim of amplifying these plasmids and making glycerol stocks. Then use the amplified IDT plasmids containing constructs B, C and D for subcloning.

Materials

Bacterial strains

For this experiment, we used the commercial strain of competent cells of E. coli JM109 from Promega, which we stored at -80°C before use.

Chemicals and Media

We used Lysogeny Broth (LB) medium, LB plates with corresponding antibiotic - ampicillin (final conc. 150 µg/ml). In addition, for transformation we used pUCIDT plasmids with our constructs B, C and D.

Short description

For this experiment, we followed the E. coli Competent Cells Quick Protocol #9FB035, which can be accessed at this website: here. To briefly introduce, competent JM109 cells stored at -80°C were placed on ice. After thawing, 100 µl of competent cells (volume of one transformation reaction) were transferred to eppendorf tubes. Subsequently, 1-50 ng of DNA pUCIDT plasmid with constructs B, C, and D (in a volume of no more than 10 µl) were added to the cells. Both positive and negative controls were also prepared to confirm that the transformation was successful and the antibiotic prevented cells without our desired antibiotic resistance from growing. Immediately after the addition of DNA, the eppendorf tubes were returned to the ice and after 10 minutes underwent a heath shock with heat in a water bath at exactly 42°C. Immediately after that, they were put back on ice again. Subsequently, LB medium was added to the transformation mixture and a 60 min incubation was performed at 37°C on a shaker at approximately 225 rpm. After one hour incubation, competent cells were diluted to 1:10 and 1:100, and plated on ampicillin-containing Petri dishes. Finally, the plates were transferred to a thermostat and incubated overnight at 37°C. The next day, the transformation was evaluated.

These cells serve as an endless source of our synthetic constructs in the form of glycerol stocks.

Subcloning of synthetic constructs to destination shuttle vector pDG3661 and pDG1664

Before transformation of JM109 cells with the destination plasmids pDG3661 and pDG1664 with our constructs, it was necessary to perform several experiments. Detailed information about these experiments can be found in the section Basic experiments and in Protocol: Isolation of plasmids.

Chronological scheme of subcloning experiments:

1. isolation of vectors pUCIDT with our constructs

2. in case of B and D construct:

   (in case of C we had to perform PCR amplification of construct from pUCIDT_C template - we were not able to digest this template successfully)

   1. restriction digestion with corresponding restriction enzymes

   2. visualization using gel electrophoresis

   3. purification

   4. ligation with appropriate destination shuttle vector

3. Transformation of JM109 cells

4. Proof of successful transformation

5. Glycerol stocks

Transformation of JM109 with destination shuttle vectors pDG3661 and pDG1664 with constructs B, C and D

Motivation

The motivation was to successfully perform several intermediate subcloning steps mentioned above and to transform E. coli JM109 with our constructs B, C and D in shuttle vectors pDG3661 and pDG1664 with the aim of amplifying these plasmids and making glycerol stocks. In addition, we want to isolate these amplified vectors in sufficient concentration to transform B. subtilis 168. The design of pDG3661 and pDG1664 vectors allows it to be subsequently integrated into a specific location of the B. subtilis 168 chromosome.

Materials

Bacterial strains

For this experiment, we used the commercial strain of competent cells of E. coli JM109 from Promega, which we stored at -80°C before use.

Chemicals and Media

As for the chemicals, we used Lysogeny Broth (LB) medium, LB plates with corresponding antibiotic - ampicillin (final. conc. = 150 µg/ml). In addition, for transformation we used shuttle vectors pDG3661 and pDG1664 with our constructs B, C and D.

Short description

Several intermediate steps that were mentioned earlier had to be taken before the actual transformation was initiated.
For transformation E. coli with pDG3661 and pDG1664 containing constructs B, C and D, we again followed the E. coli Competent Cells Quick Protocol #9FB035, which can be accessed here. The same procedure was followed as for the transformation experiment mentioned above - Transformation of JM109 with pUCIDT plasmids of our constructs B, C and D, but instead of using 1-50 ng of DNA, 10 µl of ligation mixture was used. Subsequently, the success of the transformation was confirmed by the steps presented in the following section.

Proof of transformation of Escherichia coli (JM109)

Protocol available here: Protocol: Proof of transformation of Escherichia coli

Motivation

To be sure of our success, we had to prove the presence of destination vectors pDG3661 and pDG1664 with our constructs. For this purpose, we used different methods - selection on antibiotic-containing plates, colony PCR, but also restriction digestion.

Materials

Bacterial strains

We used transformed E. coli JM109 cells.

Chemicals and Media

As for the selection, we used Lysogeny Broth (LB) medium, LB plates with corresponding antibiotic - ampicillin (conc. 150 µg/ml). For the restriction digestion, we used appropriate restriction enzymes and buffer. And sequence-specific primers, listed in the section Primers, were used to validate the colony PCR.

Short description

Firstly, the presence of the destination vectors pDG3661 and pDG1664 with constructs B, C and D in E. coli JM109 was proved by growing the cells on LB agar plates with ampicillin. We also performed better proof of transformation using restriction digestion and electrophoresis with isolated vectors from colonies of transformants to check if the sizes of the fragments were correct. Before the restriction digestion, we had to isolate plasmids from overnight culture and isolate them. You can find the detailed process of these experiments here: Basic experiments and Protocol: Proof of transformation of Escherichia coli.

Another strategy to confirm the success of the transformation is to use sequence-specific primers. The colonyPCR method can be used to amplify a specific segment of the sequence of our constructs in the destination vectors. The amplified sequence of DNA was thereafter used for various purposes including electrophoresis or storage. Further details can be found above in Basic experiments.

2. E. coli BL21(DE3)

Preparation of competent cells E. coli BL21(DE3)

Protocol is available here: Protocol: Preparation of competent cells of Escherichia coli BL21(DE3)

Motivation

To prepare competent E. coli BL21(DE3) cells to efficiently accept pUCIDT plasmids with our synthetic constructs B and D. We need to use this expression strain containing the gene for LacI in its genome to verify the functionality of our synthetic constructs B and D before and after IPTG induction.

Materials

Bacterial strains

For this experiment, we used the expression strain of competent cells of E. coli BL21(DE3), which we stored at -80°C before use.

Chemicals and Media

We used Lysogeny Broth (LB) medium, LB plates with corresponding antibiotic - ampicillin (conc. 150 µg/ml). In addition, for transformation we used pUCIDT plasmids with our constructs B and D.

Short description

After cultivation at 37 °C and subsequent inoculation, the culture was allowed to grow to an OD of 0.27-0.33 and then placed on ice for a period of time. It was now worked at 4 °C - centrifuged and a 0.05 M CaCl2 solution was added (this step was performed twice). Glycerol was added to the cell suspension. E. coli competent cells prepared in this way can be functionally stored at -70°C for approximately one year.

Testing of competent cells E. coli BL21(DE3)

Motivation

We need to verify the viability of our competent cells, which will then be transformed by plasmids with our constructs.

Materials

Bacterial strains

Expression strain of competent cells of E. coli BL21(DE3).

Chemicals and Media

We used Lysogeny Broth (LB) medium, LB plates with corresponding antibiotic - ampicillin (conc. 150 µg/ml). In addition, for transformation we used empty shuttle vectors pDG3661 and pDG1664.

Short description

E. coli BL21(DE3) cells were transformed with empty shuttle vectors pDG3661 and pDG1664 (1 µg of DNA per 100 ul of competent cells). They were plated on LB agar plates with the appropriate antibiotic, ampicillin (150 µg/ml). Untransformed competent cells plated on a plate with LB agar and ampicillin of the same concentration were used as negative control.

Transformation of competent cells E. coli BL21(DE3) with IDT plasmids (containing constructs B and D)

Protocol is available here: Protocol: Subcloning and transformation of Escherichia coli host

Motivation

We performed this experiment to deliver pUCIDT plasmids with synthetic constructs B and D into E. coli BL21(DE3) cells.

Materials

Bacterial strains

Expression strain of competent cells of E. coli BL21(DE3).

Chemicals and Media

We used Lysogeny Broth (LB) medium, LB plates with corresponding antibiotic - ampicillin (conc. 150 µg/ml). In addition, for transformation we used commercial pUCIDT plasmids containing our synthetic constructs B and D.

Short description

After thawing 200 µl of competent E. coli BL21(DE3) cells on ice, 1 µg of plasmid DNA was added. They were then left on ice for 30 min, placed in a water bath that was heated to 42 °C (heat shock) for 50 s and placed back on ice for two minutes. 1 ml of LB medium was added and left at 390 rpm for one hour at 37 °C, centrifuged and the supernatant was discarded. To the pellet of cells, 100 µl of LB medium was added and this suspension was seeded on a plate with agar and ampicillin (150 µg/ml). The dishes were retained to culture until the next day in an incubator at 37°C.

Proof of transformation of E. coli BL21(DE3)

Motivation

We need to verify which colonies grown on the antibiotic plates have pUCIDT plasmids with B and D constructs (there may be some mutant colonies on the plates that have developed resistance to the antibiotic and do not actually have pUCIDT plasmids with synthetic constructs). The verified colonies will be made into glycerol preserves and later used in further experiments.

Materials

Bacterial strains

We used transformed E. coli BL21(DE3) cells.

Chemicals and Media

As for the selection, we used Lysogeny Broth (LB) medium, LB plates with corresponding antibiotic - ampicillin (conc. 150 µg/ml). For colony PCR we used appropriate chemicals and sequence-specific primers, listed in the section Primers .

Short description

In our experiments, we used to verify the presence of plasmids with constructs in transformants in two ways. The first way is restriction analysis and another strategy to confirm the success of the transformation is to use sequence-specific primers. The colony PCR method can be used to amplify a specific segment of the sequence of our constructs in the destination vectors. The amplified sequence of DNA was thereafter used for various purposes including electrophoresis or storage. Further details can be found above in the part Basic experiments and Protocols.

Integration of construct B, C and D into the chromosome of Bacillus subtilis

Transformation of Bacillus subtilis

Our protocol for transformation of Bacillus subtilis is available here: PROTOCOL: Transformation of Bacillus subtilis

Motivation

Transformation of competent B. subtilis cells was used, because we needed to insert various vectors into our B. subtilis in order to integrate our constructs B, C and D. The first plan also included integrating construct A, sadly due to a lack of time we did not have the chance to try doing so.

Materials

We used our own naturally competent cells prepared from B. subtilis 168. The protocol for preparing B. subtilis competent cells can be found far ahead in the experiment section. We used vectors pDG1664 with construct B and pDG3661 with constructs C and D isolated from our Escherichia coli. They were carefully plated with LB medium and the corresponding antibiotic.

Short experimental description

We first had to isolate plasmids with corresponding constructs from E. coli. The naturally competent B. subtilis cells were preserved in the form of glycerol stocks. We added 1 µg of plasmid DNA to 100 µl of naturally competent cells. We also prepared positive and negative controls. The positive control contained empty supercoiled plasmid carrying antibiotic resistance. The competent cells without plasmid were used as the negative control. These controls confirmed that the transformation was performed correctly and that the antibiotic prevented cells without our desired plasmid with antibiotic resistance from growing. We incubated the tubes containing competent cells and plasmids at 200 rpm and 37 °C for 1 hour. Afterwards, we plated the competent cells on agar plates with a selection medium containing the corresponding antibiotic. The cells grew overnight at 37 °C. Next day we interpreted the results. ​​We performed the same set of experiments with pDG1664_B, pDG3661_C and pDG3661_D.

Proof of successful integration

1. AmyE test (Starch hydrolysis test)

Protocol is available here: PROTOCOL: AmyE test (Starch hydrolysis test)

Original publication:

Guérout-Fleury, A. M., Frandsen, N., & Stragier, P. (1996). Plasmids for ectopic integration in Bacillus subtilis. Gene, 180(1-2), 57–61.  https://doi.org/10.1016/s0378-1119(96)00404-0

Motivation

AmyE test is a simple method that can demonstrate ectopic integration into the chromosome of B. subtilis. 𝛼-amylase, witch degrades starch in the medium is encoded in the integration site of our plasmides. If this integration site remains intact, 𝛼-amylase is able to function normally. After the iodine treatment, this can be observed as a clear halo effect around the colonies, created by the digestion of the starch. If the plasmid with our construct is integrated successfully, 𝛼-amylase is unable to function properly and therefore cannot degrade the starch. While doing our experiments, the AmyE test was used to prove the successful integration of our inserts by comparing them with unmodified B.subtilis 168.

Materials

We used B. subtilis 168 as well as agar and starch to make the two layered agar for the plates. We also used iodine to make the digestion of starch visible.

Short description

This method utilised a two layer agar: the first is a regular agar that contains the corresponding antibiotic and the second is a thinner layer that contains starch supplement. Subsequently a few iodine crystals are added and the starch will start slowly turning dark blue. Iodine fumes are highly toxic so do not forget to work in a laboratory hood.

2. Isolation of chromosomal DNA

Motivation

Isolation of chromosomal DNA is used to confirm that our plasmids with our constructs have been successfully integrated into the DNA of B. subtilis. The isolated DNA is later used for a long PCR, where the part that we want to confirm can be amplified.

Materials

We used our modified B. subtilis 168. The full protocol can be found here: Protocol: Proof of the successful transformation of Bacillus subtilis

Short experimental description

Using an overnight culture, we used what we call a physical method which uses high temperatures to isolate the chromosomal DNA.

3. PCR

Motivation

PCR methods can amplify a certain part of DNA using the appropriate primers. We used PCR to amplify our constructs in plasmids from E. coli as well as in the chromosomal DNA of B. subtilis. Long-range PCR was routinely used in our experiment for the amplification of our longer constructs. For amplifying DNA from B. subtilis, we used colony PCR. The amplified DNA was thereafter used for various purposes including electrophoresis, DNA sequencing or storage. Further details can be found above in basic experiments.

4. Sequencing of Bacillus subtilis - amplicons of chromosomal DNA

Motivation

We needed to verify the sequence which was integrated into the chromosome of B. subtilis. This would demonstrate the success of our transformation and chromosomal integration. This experiment further demonstrates the success of our subcloning activities in B. subtilis. Few tries were needed before we achieved the final data, but the results were worth it.

Short description

We used the services of the company Eurofins Genomics for sequencing of our B. subtilis. The sequencing was performed twice. Since our targeted sequences were too long, we had to divide them into two parts. First sequenced part targeted the end of plasmid and the beginning of a construct. The second targeted part included the end of the targeted chromosome and the beginning of plasmid. We used   primers 44, 46, 51 and 52.

As far as the exact steps and methods used to prepare our cells for sequencing, we followed instructions supplied to us by the company Eurofin genomics, which can be found on their website. It is important to point out that with all three constructs: B, C and D we tried to sequence both strands of DNA using forward and reverse sequencing.

Characterization of construct B and D

Measurement of expression of fluorescent proteins

Motivation

In these experiments, we tried to demonstrate the functionalities of our project design. In our approach, we designed a reporter system to help us verify the functionality of each part of the system.

For the purpose of this experiment, we determined the growth curve based on the OD600 of our cultures. In particular, we tested the response of promoters to the presence of IPTG in the expression strain of E.coli BL21(DE3) cells. In B. subtilis cells, we were interested in constitutive expression.

We decided to verify the presence of the reporter proteins GFP and mScarlet-I in several ways. We used fluorescence measurements simultaneously with OD600 measurement of cultures on a Tecan plate fluorometer. We also used fluorescence spectrum measurements in cuvettes on a Fluoromax instrument from Horiba Scientific. Finally, we also verified the results on a fluorescence microscope.

These three experiments were performed with the same cultures. The experimental design was very complex - the cultures used, the methods, their processing and other details can be found in this table: Table of samples for measurement. In the following sections, you can read about more detailed specifications of the experiments in each facility.

1. Measurement of fluorescence and OD of culture in time - Tecan device

This device allows the measurement of fluorescence of two fluorescent proteins to be measured simultaneously. At the same time, the instrument also measures the OD of the culture, so the two types of data can be analyzed simultaneously. The instrument measures in a microtiter plate. Therefore, its use was convenient for us as it can automatically analyze a large number of samples simultaneously.

Materials

See Table of samples for measurement and other specifications. The Tecan device settings can be found in this document: Tecan settings. For the measurement, we used a black microtiter plate with 96 wells with a lid.

Fluorometer device type: Tecan Infinite 200 Pro

Method

This machine allows you to measure samples in a microtiter plate. The instrument maintains the culture temperature at 37 °C and also provides shaking of the microtiter plate. Measurements of OD, GFP intensity and mScarlet-I were performed automatically at 15 min intervals over a 24 h period. A bottom beam was used to measure fluorescence intensity and OD. This method of measuring from underneath is well proven and more accurate in the laboratories where we made the measurements. For higher reproducibility of the results, we worked in technical duplicates.

Preparation of microtiter plate lid

We used a black microtiter plate with a lid for 96 samples. 0.05% Triton in ethanol was poured onto the lid. Care must be taken to ensure that the entire surface of the cap is coated with an even thin layer. Then the lid is left inside up, the flowbox is closed and the plate and lid are illuminated with UV light. This ensures the sterility and cleanliness of the plate. The plate should also be sterilized with UV light before each subsequent use. For subsequent use, the sample can only be placed in the unused wells only.

Sample preparation for measurement

Overnight cultures in liquid LB were prepared the previous evening (culture at 110 RPM shaking, 37 °C). The following morning their OD was measured. Subsequently, the cultures were diluted appropriately so that their OD was 0.05. 200 ul of each culture was pipetted into a well in the plate. Once the OD of the samples reached 0.6, the BL21(DE3)_B IPTG 0.6, BL21(DE3)_D IPTG 0.6 and BL21(DE3) IPTG 0.6 samples were induced with IPTG to a final concentration of 100 uM. The B. subtilis positive control GFP in chromosome sample was also induced at OD = 0.6, but by adding xylose to a final concentration of 0.5%. See the supplementary chart.

2. Measurement of fluorescence in device FluoroMax Plus

The spectrofluorometric measurement is an essential confirmation that the correct fluorescent proteins are formed in our constructs. It can very accurately measure the excitation and emission spectra of any fluorescent protein. However, this instrument is very computationally expensive, so we are grateful to the management of Loschmidt Laboratories for allowing us to make measurements on it.

Material

For this experiment, we used a similar set of samples as for the previous measurements in the Tecan device. The list of samples used can be found in this document: Table of samples for measurement. Cultures were not diluted before measurement. Each sample was measured in a polystyrene disposable 4 sided cuvette.

Preparation of samples for measurement

Overnight cultures of all samples in LB medium were inoculated the previous day (shaking culture at 110 RPM, 37 °C). Measurements were performed 20 hours after inoculation of overnight cultures. Certain cultures were induced with IPTG immediately after inoculation. Some samples were diluted after 15 hours of cultivation to OD = 0.8. Some samples were induced at OD = 0.8 by IPTG to a final concentration = 100 uM and these were returned to the shaker. For other samples, a double concentration of IPTG was added.

Method:

The type of device: FluoroMax Plus TCSPC, Horiba Scientific

Other device specifications:

• xenon lamp,

• monochromatic excitation = diffraction grating,

• the reference detector = photodiode,

• the emission monochromator = diffraction grating,

• the main detector = PMT,

• slit = 1 nm for excitation and emission

We used the Horiba Scientific FluoroMax Plus instrument provided by Loschmidt Laboratories for this experiment, to whom we are very grateful. Spectral analysis of the emission spectra of each sample was performed. There was a range of wavelengths for mScarlet-I and GFP which you can see in the following table:

Measured fluorescent protein	Setting the range of spectra
GFP	400 - 600 nm
mScarlet-I	500 - 700 nm

Table 2 : Measurement specifications of FluoroMax Plus

Fluorescence microscopy

We aimed to verify the measurements made on the previous instruments using a fluorescence microscope. Selected cultures were photographed in a brightfield and using fluorescence filters.

Material:

We used some of the cultures prepared for the previous two experiments - which exactly is indicated in the Table of samples for measurement. For less light refraction, immersion oil was used.

Method:

For this experiment, we used this type of fluorescence microscope: Imager Z2, Zeiss.

The used microscope and other specific device settings can be seen at the two following pictures:

15 ul of culture were taken and mixed with 15 ul of immersion oil. The sample prepared was thus ready for photography. A drop of immersion oil was added to the top of the coverslip before photographing. The appropriate filters and magnification were set on the microscope. Each specimen was attempted to be captured with either brightfield, GFP and mScarlet-I filters.

Igem Team Brno, Czech Republic 2021