Results

Introduction

The synthetic sequences as well as regulatory regions used in our project PHOSCAGE were designed using the Benchling software. In silico, 4 gene constructs were designed, called as constructs A, B, C and D.

Construct A was designed to test BMC production. However, this construct could not be synthesized by IDT and hence will not be mentioned in the following sections.

Construct B contains a gene for sfGFP production with a PduP tag that should ensure sfGFP packing to BMCs.

Construct C contains genes for the cI repressor and mScarlet-I downstream of the P_Pho promoter. This construct is designed to test the response of P_Pho to changes in phosphate levels.

Construct D is responsible for the formation of cI repressor and mScarlet-I with a degradation tag upon IPTG induction. In the absence of IPTG, GFP with a degradation tag is in turn expressed.

More detailed information about our constructs can be found in Parts and Project design.

Each construct contains BamHI and HindIII restriction sites at the 5' and 3' end respectively. Corresponding restriction enzymes were used to clone each construct into its destination integration plasmid from which it can be integrated into the specific locus of B. subtilis 168 chromosome.

Synthetic sequences (Parts) received from the company IDT were delivered in pUCIDT plasmids. For this reason, we used a technique known as subcloning. This technique is used for transferring a specific DNA sequence from a parent vector, in our case the commercial plasmid pUCIDT, to a target vector. Target vectors used in our project were plasmids pDG3661 and pDG1664. Both of them are “shuttle vectors”, which means that they can be used in both E. coli and B. subtilis.

After successful transformation of E. coli JM109 with pUCIDT_B, pUCIDT_C, and pUCIDT_D, these plasmids were isolated from overnight cultures, digested by corresponding restriction enzymes, and ligated to the shuttle vector pDG3661 (in the case of constructs C and D) or pDG1664 (in the case of construct B). The destination vectors thus ligated with constructs B, C and D (pDG3661_C, pDG3661_D, pDG1664_B) were again used for the transformation of E. coli JM109 which was then utilised for their replication. All thusly prepared vectors were isolated from overnight cultures. B. subtilis 168 was transformed with these vectors and our constructs and parts of plasmids were integrated into its chromosome. The functionality of synthetic systems B and D was subsequently tested.

After the first testing measurements, the E. coli strain BL21(DE3) was also transformed with pUCIDT_B and pUCIDT_D and the functionality of the synthetic constructs was tested in this expression strain with LacI repressor gene in its genome (Schema of our experimental approach is shown in Figure 1).

In the following section you will find detailed information about results of all the experiments performed.

Preparation of Escherichia coli cloning host

Experimental design

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Figure 1:Scheme of our experimental approach.

Construct B was cloned into the destination pDG1664 vector by restriction digestion and ligation - creating pDG1664_B which was then used to transform E. coli JM109. Transfromants were selected by their ampicillin resistance. Constructs C and D were cloned into the destination vector pDG3661 - creating pDG3661_C and pDG3661_D. After transformation of E. coli JM109, ampicillin was also used for selection. We were able to clone constructs B and D into the destination vectors without too much trouble. However, in the case of construct C, this was more complicated and therefore a PCR cloning method was used instead of subcloning.

In order to perform complex measurements, which are described in the last section of “Wet lab and Results” , the constructs were cloned not only into cloning strain E. coli JM109, but also into expression strain E. coli BL21(DE3).

At the end of the section “Preparation of Escherichia coli cloning host” , we have isolated the vectors pDG3661_D, pDG3661_C and pDG1664_B in high concentrations. The success of cloning and subsequent transformation of JM109 and BL21(DE3) was demonstrated by plasmid isolation, colony PCR or control restriction digestion.

Transformation of E. coli JM109 with pUCIDT_B, pUCIDT_C and pUCIDT_D

All of our constructs were synthesized by the IDT company and delivered in pUCIDT vectors. pUCIDT_B, pUCIDT_C and pU CIDT_D were successfully used for transformation of E. coli JM109 competent cells (Figure 2 and 3) from which cell glycerol stocks were prepared. The transformation is described in the PROTOCOL: Subcloning and transformation of Escherichia coli host. The pink coloration of some colonies caused by mScarlet-I production in the Figure 3 is a strong indication of successful transformation of E. coli JM109 with pUCIDT_C. The colonies transformed with pUCIDT_D were more orange in colour, probably resulting from the combination of red and green fluorescent proteins. This therefore suggests that expression of our constructs is taking place in E. coli in spite of the fact that the constructs are designed and optimized for B. subtilis.

Figure 2: Transformation of E. coli JM109 competent cells with pUCIDT_B. Plate number 2 is vital control for competent cells. Plate number 3 contains transformed cells with vector pUCIT_B and number 4 contains negative control. Plate number 1 is a test of our antibiotic stock, which is not significant for this experiment.

Figure 3: Transformation of E. coli JM109 with pUCIDT_C and pUCIDT_D. Plate 1 and 2 are vital controls for competent cells, plate 3, and 5 are cells transformed with pUCIDT_C and 4 and 6 are cells transformed with pUCIT_D. Plate number 7 is negative control.

A problem arose in the synthesis of our construct A, which required a design change. pUCIDT_A was therefore not delivered to us until late summer and thus we were not able to start working with this construct before the end of the competition.

Proof of transformation of E. coli JM109 by pUCIDT_B, pUCIDT_C and pUCIDT_D

Successful transformation of the vector pUCIDT_B was confirmed by selection on plates containing ampicillin. Transformation was also verified by restriction digestion with the enzymes HindIII and BamHI. Size of the cleaved construct (1128 bp) and vector (2770 bp) was determined using gel electrophoresis and a gene marker see Figure 4. Fragments of construct B (marked by arrow) were extracted from gel and also used for ligation with digested vector pDG1664 described in the next section.

Figure 4:Proof of transformation of E.coli JM109 - restriction analysis.DNA fragments from restriction digestion of pUCIDT_B isolated from E.coliJM109 demonstrating successful transformation. M is marker - GeneRuler 1 kb (ThermoFisher Scientific). Samples 1 and 2 are pUCIDT_B digested with BamHI and HindIII - fragments labeled by arrow had the length of digested construct B - 1128 bp and vector 2770 bp. Electrophoresis conditions: 0,8% agarose gel, 120V, 45 min.

Figure 5: Restriction digestion of pUCIDT_C and pUCIDT_D plasmid as a proof of successful transformation.DNA fragments obtained by restriction digestion of pUCIDT_C and pUCIDT_D proved successful transformation of JM109 E.coli cells. M is marker - GeneRuler 1 kb (ThermoFisher Scientific), First sample to the right is digested pUCIDT_C 2295 bp long. Next sample is the vector pDG3661 (which was used for subsequent ligation) digested with BamHI and HindIII. The last sample is pUCIDT_D digested with BamHI, HindIII and BsaI where the size of the digested construct was 2891 bp. Electrophoresis conditions: 0,8% agarose gel, 120V, 45 min.

Successful transformation of E. coli JM109 with pUCIDT_C and pUCIDT_D was verified in the same manner. After restriction digestion, construct C had 2295 bp and construct D 2891 bp, see Figure 5.

Construct D was similar in size to the digested empty pUCIDT, so pUCIDT_D had to be additionally digested with enzyme BsaI, resulting in two plasmid fragments of sizes 1355 bp and 1415 bp (which appear as a single band on the gel). This allows for nice separation of digested construct D and pUCIDT plasmid and thus improves gel extraction.

Cloning of pDG1664_B, pDG3661_C and pDG3661_D and transformation of E. coli JM109

After previously described restriction digestion, gel electrophoresis and gel extraction, construct D was successfully ligated with shuttle vector pDG3661 and used for the transformation of E. coli JM109.

Attempts at ligating construct C with this vector resulted in repeated contamination with the original pUCIDT vector. pUCIDT_C was likely to have a significantly better transformation rate or better ability to replicate in competent cells. This problem was most likely the result of choosing the same selection marker, i.e. ampicillin resistance, for both vectors. This problem was eventually solved by amplifying construct C with PCR (primers marked as 55 and 56 can be found here, the size of the amplicon was 2471 bp) and then ligating the amplicons with vector pDG3661. Products of this ligation reaction were then successfully used for the transformation of E. coli JM109.

After subcloning our constructs into shuttle vectors pDG3661 (C and D) and pDG1664 (B) the cloning and transformation was verified as described below (see Figure 6, Figure 7, Figure 8 and Figure 9).

Figure 6:Transformants of E. coli JM109 containing pDG1664_B on plates with ampicillin.Plate 1 contains negative control. Plate 2 contains positive control. Plates 3 and 4 were inoculated with cells transformed by pDG1664_B from a ligation mix marked as B2, these two plates differ by the amount of inoculum. Plates 5 and 6 contain cells transformed by pDG1664_B from a ligation mix marked as B1, these two plates also differ by the amount of inoculum.

Figure 7 : Transformants of E. coli JM109 containing pDG3661_C on plates with ampicillin.Plates 1 and 3 contain cells transformed with a ligation mix marked as C1. The difference between them is in the amount of inoculum (400 µl for the plate 1 and 110 µl for the plate 3). Plates 2 and 4 contain cells transformed with ligation mix marked as C6. The difference between them is in the amount of inoculum (400 µl for the plate 2 and 110 µl for the plate 4). Plate 5 is positive control containing cells transformed with plasmid pDG3661 and plate 6 is negative control.

Figure 8: Transformation of E. coli JM109 containing pDG3661_D on plates with ampicillin.Plate 1 is vital control of our competent cells on medium without ampicillin. Plate 2 is positive control and plate 3 is negative control. Plate 4 contains transformants with pDG3661_D from the ligation mix marked as D2 (colony marked with an arrow). Plate 5 contains transformants with pDG3661_D from the ligation mix marked as D1 (colony marked with an arrow).

Figure 9: Transformation of E. coli JM109 containing pDG3661_D on plates with ampicillin.Figure 9 is a close up of the figure 8 for better visibility of our colonies (marked with arrows).

Proof of transformation of E. coli JM109 by pDG1664_B, pDG3661_C and pDG3661_D

Successful transformation of E. coli with pDG1664_B was verified by restriction digestion with BamHI and HindIII (Figure 10). Fragment size of the construct remains 1128 bp, the size of digested pDG1664 plasmid is 6042 bp.

Figure 10: Proof of transformation of E.coli JM109 by pDG1664_B - restriction analysis.This figure shows DNA fragments from restriction digestion of pDG1664_B which were isolated from E.coli JM109 demonstrating successful transformation. M is marker - GeneRuler 1 kb (ThermoFisher Scientific). First three samples (1 - 3) are pUCIDT_B digested with BamHI and HindIII fragments had the length of digested construct B - 1128 bp and vector - 2770 bp. Samples 4-6 are pDG1664_B digested with BamHI and HindIII fragments had the length of digested construct B - 1128 bp and vector - 6042 bp. Electrophoresis conditions: 0,8% agarose gel, 120V, 40 min.

Successful transformation of pDG3661_C into E. coli was confirmed by colony PCR (primers 1 and 2 were complementary to pDG3661_C, primers 55 and 56 were complementary to pUCIDT_C and were used to detect potential contamination by this plasmid - they can all be found here). As can be seen on the gel in Figure 11, some pUCIDT contamination was still present. The colony which contained only the plasmid pDG3661_C (amplicon of the size 2570 bp) was marked as number 1 and was used for subsequent experiments. Shuttle vector containing construct C was isolated from colony 1 and could finally be used for the transformation of B. subtilis , where it was integrated into its chromosome (described in Transformation of Bacillus subtilis below).

Figure 11: Colony PCR proving successful transformation of E. coli with pDG3661_C. M is marker - GeneRuler 1 kb (ThermoFisher Scientific).The numbers (1, 2, 3, 4) indicate the number of the colony which was sampled for the cPCR. The letters (a and b) indicate the combination of used primers (a = primers 1 and 2 - to identify pDG3661_C, b = primers 55 and 56 - to identify pUCIDT_C). In the sample 1a was confirmed pDG3661_C (size of the amplicon was 2570 bp, marked by an arrow). Sample 1b contains a non-specific product, sample 2a contains a non-specific product (pDG3661_C not present), 2b shows the presence of pUCIDT_C and non-specific products. In 3a, no products were detected. 3b shows the presence of pUCIDT_C and a non-specific product. Electrophoresis conditions: 1% agarose gel, 120V, 40 min.

Successful transformation of E. coli with pDG3661_D was demonstrated by restriction digestion. Fragment size of construct D was 2955 bp and 7271 bp for plasmid pDG3661, see Figure 12.

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Figure 12: Proof of cloning construct D into the shuttle vector pDG3661 andof successful transformation of E. coli : M is marker - GeneRuler 1 kb (ThermoFisher Scientific).Based on the size of the fragments (2295 bp for construct C and 2768 bp pUCIDT) from restriction digestion were samples C1 and C2 identified as pUCIDT_C. Samples D1 and D2 contain pDG3661_D (2955 bp for construct D and 7271 bp for pDG3661). PC = positive control (pDG3661), NC = negative control. Electrophoresis conditions: 1% agarose gel, 120V, 40 min.

Transformation of competent E. coli BL21(DE3) with pUCIDT_B, pUCIDT_D

We have successfully integrated our constructs B and D into the B. subtilis 168 chromosome (described below), but after testing their exprethis was a result of the absence of the LacI repressor gene in the genome of B. subtilis 168. As there is nothing to repress IPTG inducible promoters in the absence of IPTG, the expression of genes under their control is constitutive.

We therefore decided to also transform E. coli BL21(DE3) expression strain, which contains the gene for LacI in its genome, to demonstrate the response of our system to IPTG induction.

Plasmids pUCIDT were used for the transformation of E. coli BL21(DE3) as they are smaller than the shuttle vectors pDG3661 and pDG1664 and are present in E. coli cells in more copies. This should ensure a high rate of production of target proteins.

After testing competent E. coli BL21(DE3) cells, we transformed them with pUCIDT_B (see Figure 14) and pUCIDT_D (see Figure 13). Both transformations were successful. As a negative control, we used competent cells without plasmids plated on a Petri dish with ampicillin (150 µg/ml). The verification of successful transformation and its outcome will be described in the following section.

Figure 13: Colonies of E. coli BL21(DE3) competent cells transformed with pUCIDT_D. Plate 1 - 1x diluted, plate 2 - 10x diluted, plate 3 - 100x diluted.Plates contain ampicillin.

Figure 14: Colonies of E. coli BL21(DE3) competent cells transformed with pUCIDT_B. Inoculate on plate 1 was 10x diluted. Inoculate on plate 2 - 100x diluted.Plates contain ampicillin.

Proof of transformation of E. coli BL21(DE3) with pUCIDT_B and pUCIDT_D

First preliminary indication of successful transformation was the growth of colonies on our ampicillin (150 µg/ml) plates. Colony PCR was also used for additional verification of the transformants. Sequence-specific primers 55 and 56 annealing to pUCIDT were designed to be used with all constructs ligated to pUCIDT plasmids. All primers can be found in the Primers section. Successful transformation of E. coli BL21(DE3) with pUCIDT plasmids containing constructs B and D was thus confirmed. pUCIDT plasmids with the respective constructs isolated from our stocks of transformed E. coli JM109 were used as positive controls. In negative control, 1 µl of PCR water was added to the PCR reaction instead of DNA. Electrophoresis was performed at 120 V for 33 min and the products were analyzed on a 0.8% agarose gel.

The products of the PCR reactions had the expected size in both cases. The gel in Figure 15 demonstrate successful transformation of 2 colonies with pUCIDT_B and two colonies with pUCIDT_D. Later on, more colonies were tested and glycerol stocks were made from the cells of verified colonies.

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Figure 15: PCR products proving the successful transformation of E. coli BL21(DE3) with pUCIDT_B and pUCIDT_D.First sample from the left (NC) is the negative control. Samples D1 and D2 are products of PCR reaction which confirm the presence of pUCIDT_D in tested colonies (size of the amplicon is 3070 bp). Sample D2 also contains a non-specific product. PC-D is positive control (isolated pUCIDT_D from previous experiments).Samples B1 and B2 are products of PCR reaction which confirm the presence of pUCIDT_B in tested colonies (size of the amplicon is 1275 bp). (PC-B) is positive control (isolated pUCIDT_B verified in previous experiment). M is marker - GeneRuler 1 kb (ThermoFisher Scientific). Electrophoresis conditions: 1% agarose gel, 120V, 40 min.

Integration of synthetic constructs into the chromosome of Bacillus subtilis

Experimental design

In the next phase of our work in the wet lab, we focused on transforming Bacillus subtilis strain 168 with plasmids pDG1664_B, pDG3661_C and pDG3661_D. Working with bacteria B. subtilis was a challenge, since there was no one at our University with previous experience with this bacterium except from our 2020 iGEM team. Therefore this project was a challenge for us as well as our PIs at times. Despite this, we have managed to perform our experiment successfully and finish as planned with the exception of a few time management flaws.

We are very thankful for the clever and insightful advice we received from Dr. Krásný and Laboratory of Microbial Genetics and Gene Expression in Prague. Their help is greatly appreciated.

The vectors pDG1664 and pDG3661 were designed for the integration into the chromosome of B. subtilis . The part of pDG1664 designed for integration additionally encodes resistance to erythromycin and the combination of erythromycin and lincomycin was used for the selection of transformants containing pDG1664_B. The part of pDG3661 designed for integration carries resistance to chloramphenicol. AmyE test was used to demonstrate successful transformation and integration of pDG3661_C and pDG3661_D into the AmyE region of the chromosome B. subtilis 168. Additionally, chromosomal DNA was isolated and the region of insertion was amplified by PCR. PCR products were then sent for sequencing.

Testing competent cells of Bacillus subtilis

We worked with competent cells of B. subtilis 168 which we obtained last year from Dr. Krásný from Prague. Before using them, we first decided to test them and see if they are still functional. To make sure that they are still ready to be transformed, we transformed them with empty vectors pDG3661 and verified the integration into the chromosome by PCR. The PCR showed that the cells were still competent.

Transformation of Bacillus subtilis with pDG1664_B, pDG3661_C and pDG3661_D

More details about our experiments can be found in the section Wet lab.

As the synthesis of some of our composite parts took longer than others and some trouble arose during cloning and transformation of certain constructs, we were not able to work with all of our constructs simultaneously, though we tried to work in parallel when possible. The first successful transformation and integration into the chromosome of B. subtilis was performed with Construct D. Then integration of construct B was close second. Working with construct C however took substantially longer due to the difficulties with subcloning into pDG3661. Transformed strains of B. subtilis 168 were named B. subtilis_B , B. subtilis_C and B. subtilis_D , according to the construct which was integrated into their chromosome.

Transformed cells were streaked onto plates with the appropriate antibiotic and incubated overnight at 37°C. In the figure 16 below you can see plates with B. subtilis transformants carrying construct C.

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Figure 16: Transformants of B. subtilis 168 with pDG3661_C integrated in its chromosome on plates with chloramphenicol.Plates 1, 2 and 3 contain transformants with pDG3661_C. In this case, positive control did not grow. The transformation with pDG3661_C was however tested and confirmed by PCR and sequencing. Plate 5 contains negative control. The fact that colonies can be seen on negative control is probably due to spontaneous mutations leading to resistance.

Proof by PCR and agarose gel DNA electrophoresis

First step of verifying the integration of our constructs into the chromosome of B. subtilis 168 was long PCR using primers which annealed either near the integration site on chromosome of B. subtilis or to our composite part. Chromosomes isolated from transformed colonies were used as templates for these PCR reactions. Chromosomes were isolated using a “physical method” of isolation which utilizes the alternation of extreme temperatures to break the cell wall. More details can be found in the section Wet Lab.

For constructs B and C, only the region encompassing the transition between the chromosome of B. subtilis , the part of plasmids pDG1664 and pDG3661 designed for integration and the 5' end of our construct were amplified. For chromosomes containing construct B, primers 43 and 46 were used to amplify the transition between chromosome and plasmid (685 bp) and primers 50 and 51 were used to obtain a PCR product encompassing the transition between plasmid and our construct (935 bp). For chromosomes containing construct C, primers 44 and 46 were used to amplify the transition between chromosome, plasmid and our construct (1117 bp).

For construct D, The entire inserted region was amplified using primers 28 and 29, which anneal to the chromosome of B. subtilis at each side of the integration site, producing an amplicon of 6511 bp.

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Figure 17: PCR verification of successful integration of construct B intochromosome.For sample 2 primers 43 and 46 were used and the amplicon of 685 bp was produced. For sample 1 primers 50 and 51 were used and the size of the amplicon was 935 bp. The sequence of sample 2 in the second well was also verified by sequencing (see below). M is marker - GeneRuler 1 kb (ThermoFisher Scientific). Electrophoresis conditions: 1 % agarose gel, 120V, 40 min.

Figure 18: PCR verification of successful integration of construct D intochromosome of B. subtilis 168. M is marker - GeneRuler 1 kb (ThermoFisher Scientific).Primers 28 and 29 were used for samples D1 and D2. The size of the product should be 6511 bp. Amplicon of this size is only present in sample D1. Positive control (PC) is a sample of B. subtilis with plasmid pDG1664 inserted in its chromosome. NC is a negative control. Electrophoresis conditions: 1 % agarose gel, 120V, 40 min.

Figure 19: PCR verification of successful integration of construct C intothe chromosome of B. subtilis 168. PCR products amplified from the B. subtilis_C chromosome were loaded on the gel.Primers 44 and 46 were used to amplify the region of transition between the chromosome and the insert. Vector pDG3661_C was used as a positive control with primers 52 and 53. The size of the amplicons (1117bp) confirms the presence of the insert in the chromosome of B. subtilis . The amplicon of the positive control is also of the correct size - 900 bp. The same samples were then extracted from the gel and sent for sequencing (see below). NC = negative control. M is marker - GeneRuler 1 kb (ThermoFisher Scientific). Electrophoresis conditions: 1 % agarose gel, 120V, 40 min.

Proof by AmyE test

Another proof of successful integration of plasmid pDG3661 into the amyE region of the chromosome of B. subtilis 168 is the AmyE test. In the case of construct B, the insert is not integrated into amyE region but into thrC so AmyE test would not work. More details about this experiment can be found in the section Wet Lab.

The ability of transformed cells to digest starch is compared with cultures of unmodified B. subtilis 168 strains. The cells are grown on plates containing starch. After some time of incubation, iodine is added onto the plates. If the cells are able to metabolize starch, iodine will not stain the solid medium in their proximity, forming a sort of halo. As the amyE gene which is necessary for starch digestion is inactivated by the insertion of pDG3661 into its sequence, no halo should be present in cell cultures with successfully integrated pDG3661_C and pDG3661_D. The AmyE test confirmed successful integration in all tested colonies (see Figure 20).

Figure 20: AmyE tests. 1.: Unmodified B. subtilis - negative control, 2: B. subtilis_D 3: B. subtilis_C .Typical halo around the lawn of unmodified B. subtilis can be seen on starch plate 1, demonstrating the functionality of amyE gene of our negative control. On starch plates number 2 and 3, no halo can be seen around the lawn of B. subtilis . This demonstrates successful insertion of pDG3661_C and pDG3661_D into amyE locus responsible for starch digestion.

Proof by sequencing

Firstly, the chromosomal DNA of B. subtilis_B, B. subtilis_C and B. subtilis_D had to be isolated using the previously mentioned “Physical method” which utilises extreme temperatures to break the cell wall.

As our constructs are rather long, we decided to only send the transition between the chromosome and the part of the integrated plasmid and the transition between the integrated part of the plasmid and our construct for sequencing as these sequences were crucial for demonstrating successful integration. The selected sequences were first amplified by long PCR. The PCR product was then separated from the rest of the chromosomal DNA on agarose gel DNA. The correct amplicon was extracted from the gel and the correct amount of DNA with a corresponding primer (see Table of primers) was sent for sequencing.

In the case of B.subtilis_D we used primers 44 (transition from chromosome to plasmid) and 52 (transition from plasmid to construct D), for B we used 46 (transition from chromosome to plasmid) and 51 (transition from B to plasmid) and for C we used 47 and 44. We prepared samples according to the instructions from Eurofins corporation.

The alignments of our sequences can be found here:

B.subtilis_D_Primer44 ,B.subtilis_D_Primer52

B.subtilis_C_Primer44 ,B.subtilis_C_Primer47

B.subtilis_B_Primer46 ,B.subtilis_B_Primer51

The integration of constructs B, C and D into the chromosome of B. subtilis 168 was successfully demonstrated by sequencing. The schemes below show sequenced parts of integration regions of B. subtilis chromosome - B. subtilis_B (Figure 21), B. subtilis_C (Figure 22) and B. subtilis_D (Figure 23).

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Figure 21: Scheme of sequenced part of integration region of B.subtilis_B chromosome.

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Figure 22: Scheme of sequenced part of integration region of B.subtilis_C chromosome.

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Figure 23: Scheme of sequenced part of integration region of B.subtilis_D chromosome.

Characterization of constructs B and D

Measurement of expression of fluorescent proteins

In our PHOSCAGE project, four gene constructs - A, B, C and D - were designed. Following experiments were performed with constructs B and D.

Construct B contains a gene encoding sfGFP (Superfold GFP) with a PduP tag that should ensure its encapsulation into BMCs (production of which is governed by construct A).

Construct D is responsible for the expression of cI repressor and mScarlet-I with a degradation tag upon IPTG induction. In the absence of IPTG, GFP with a degradation tag is expressed in turn.

More detailed information about our constructs can be found in Parts and Project design.

Experimental design

After successful transformation of E. coli and B. subtilis , we designed the following experiments to demonstrate the functionality of the different components in our project. We included a reporter system in our design to more easily verify and visualize the functionality of each component of the system.

First, we determined the growth curve for each culture of E. coli and B. subtilis recombinants from regular measurements of OD600. We tested how the system responds to the presence of IPTG in the culture of the expression strain E.coli BL21(DE3). In B. subtilis cells, we presumed that IPTG inducible promoters would behave as constitutive.

We used several different methods to detect the presence of the reporter proteins GFP and mScarlet-I. We used a Tecan Infinite 200Pro plate spectrophotometer for simultaneous and continuous measurement of both OD600 and fluorescence of the cultures. Additionally, we used FluoroMax Plus from Horiba Scientific for more sensitive measurement of fluorescence of B. subtilis and E. coli cells in cuvettes in end point mode. We then verified the results obtained using these two techniques by fluorescence microscopy.

All the experiments were performed with the same cultures. The experimental design was complex and more details about the cultures used, courses of the cultivation and more can be found in this table: Table of samples for measurement. Following pictures (Figures 24, 25 and 26) were taken during sample preparation.

Figure 24: Presence of fluorescent protein in liquid cultures of strainscarrying constructs B and D. Cultures of E. coli BL21(DE3) transformed with constructs B (left flask) and D (two flasks on the right).The presence of matured fluorescent protein is clearly visible after prolonged cultivation even to the naked eye.

Figure 25 : Liquid cultures of strains carrying constructs B and D under UV light. After prolonged cultivation, cultures containing our constructs were placed under UV light. The culture of E. coli BL21(DE3)_pUCIDT_D on the bottom left shows bright pink fluorescence. The culture at the bottom right contains B. subtilis strain from Dr. Krásný which was transformed with the gfp gene using plasmids. This culture glows with hints of green fluorescence. We planned on using this as a positive control, but ended up using a different strain instead. The two cultures at the back are the negative controls - B. subtilis 168 and E. coli DH5𝛂 which do not carry any genes encoding fluorescent proteins.

Figure 26: BL21(DE3) colonies of strains carrying constructs B and D onplates under UV light.BL21(DE3) colonies with strains transformed with our constructs were streaked on LBA plates with corresponding antibiotics. The colonies displaying green fluorescence on the left are of the strain BL21(DE3)_pUCIDT_B . The plate below is the BL21(DE3) control which was not transformed with any of our constructs. On the right are BL21(DE3)_pUCIDT_D colonies, glowing bright pink due to mScarlet-I expression. The plate below is the control. Plates were incubated for 16 hours at 37 ͒C.

As demonstrated in figures 24 and 25, the presence of fluorescent proteinsin the cells carrying our constructs is visible to the naked eye both under UV light and daylight after prolonged cultivation. This observation corresponds to the results obtained from further measurements.

BL21(DE3) cells depicted in figure 26 were plated on petri dishes with LBmedium and appropriate antibiotics. Unlike the controls (two plates at the bottom), the colonies carrying construct B (on the left) and D (on the right) showed fluorescence of corresponding colour.

1. Continuous measurement of OD and fluorescence in liquid cultures with recombinant Bacillus or E. coli using Tecan spectrophotometer.

This device is able to simultaneously measure the fluorescence of two different fluorescent proteins as well as the OD of the culture, so the data obtained can be analyzed together. The parameters were measured in cultures incubated in a microtiter plate.

For a more detailed description, see the section Characterization of construct B and D on the page Wet lab. Sample selection and preparation can be found in the following table: Table of samples for measurement.

Following measurements were all conducted during one run of the experiment in the Tecan spectrophotometer. Figure 27 shows the growth curves of E. coli BL21(DE3) strains carrying constructs B or D as well as controls that were not transformed with our constructs. Some of the cultures were induced with IPTG during inoculation, some were induced at OD 0.6 and some were not induced.

Figure 27: Growth curves of E. coli BL21(DE3) cultures used in following experiments.The graph shows the growth curves of selected strains of Escherichia coli BL21(DE3). The different strains are labeled in figure legend. The designation "_pUCIDT_B/D" signifies the presence of our constructs in pUCIDT plasmid. The label "IPTG" indicates the presence of the inducing agent and the label "0.6 / overnight" indicates the point of induction. The black curve is the measurement of the blank.The blue dashed line indicates the time of induction at OD 0.6.

The decrease in growth rate of cultures after induction indicates a slighttoxic effect of IPTG. Even the control culture which was not transformed with our construct and was induced overnight ( BL21(DE3) IPTG overnight ) was negatively affected by IPTG as it grew slower compared to control BL21(DE3) without IPTG where IPTG was not added.

Interestingly, the growth curve of strains with construct B - BL21(DE3)_pUCIDT_B IPTG overnight and BL21(DE3)_pUCIDT_B IPTG 0.6 did not show significant differences after induction compared to the cultures of cells carrying construct B which were not induced. The growth curve of these cultures was similar to the controls. This could imply that the expression of construct B does not pose a metabolic burden on the BL21(DE3) cells.

The presence of construct D, on the other hand, poses a great metabolicburden on the BL21(DE3) cells. The lag phase of cultures transformed with construct D was prolonged by more than 1 hour.

Figure 28 shows the growth curve of B. subtilis strains used in following experiments. We analyzed strains containing constructs B or D, negative controls which were not transformed with our constructs and also a positive control with GFP gene integrated in its chromosome. The positive control with integrated GFP gene was induced at the same time as BL21(DE3) cultures as both experiments were performed simultaneously.

Figure 28: Growth curves of B. subtilis 168 cultures used in following experiments.The graph shows the growth curves of selected strains of Bacillus subtilis. The different strains are labeled in the annotation of the graph. The label "B/D" signifies the presence of our construct. The black curve is the measurement of the blank (LB medium). The blue dashed line indicates the time of xylose induction of B. subtilis positive control GFP in chromosome.

These results are in accordance with predicted behaviour of selected B. subtilis cultures. Bacillus with constructs B or D integrated into their chromosome as well as cultures where only a part of the empty plasmid was integrated grew more slowly in the late growth phase.

Simultaneously with OD, relative fluorescence of these cultures was also measured. Figure 29 shows the measurement of mScarlet-I fluorescence of E. coli BL21(DE3) cultures in time. Measured were strains with construct D inserted into their chromosome as well as control strains with no integration - BL21(DE3) without IPTG and BL21(DE3) IPTG overnight . Some samples were induced with IPTG already at inoculation and one was induced at OD 0.6. The exact instrument settings for this type of measurement can be found in the section: Wet lab, Characterization of construct B and D.

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Figure 29: Fluorescence of mScarlet-I in cultures of E.coli BL21(DE3) strains carrying construct D and negative controls measured in time.The graph displays the fluorescence values in Relative Fluorescence Unit (RFU) of mScarlet-I of selected strains of Escherichia coli BL21(DE3) measured in time. The different strains are labeled in the annotation of the graph. The label "IPTG" indicates the presence of the inducing agent and the label "0.6/overnight" indicates the time of induction. The black curve is the measurement of the blank.The blue dashed line indicates the time of induction.

No increase in fluorescence was detected in the blank nor in the negativecontrols. The results show an increase in fluorescence of strains with integrated construct D when compared to the controls. Sharp increase in fluorescence was however recorded only after a longer period of time. IPTG induction at OD 0.6 had no effect on the intensity of fluorescence. The increase in intensity of fluorescence of the BL21(DE3)_pUCIDT_D IPTG overnight was delayed.

Unfortunately, we did not have the time to optimize the settings of GFPfluorescence measurement in the Tecan fluorometer and thus the results we obtained so far were inconclusive and are not presented here.

2. Fluorescence measurements in FluoroMax Plus and fluorescence microscopy of Bacillus or E. coli recombinants

Spectrofluorimetry was used to confirm correct folding and expression of fluorescent proteins in recombinant strains cultured in liquid media. Horiba Scientific FluoroMax Plus can very accurately measure the emission spectra of our fluorescent proteins. Range of measured spectra displayed in the figures below cover the emission peaks of GFP and mScarlet-I.

Other device specifications are listed on the page Wet lab in the section Characterization of project design in case of construct B and D.

a) The effects of IPTG induction on fluorescence intensity in E.coli BL21(DE3)

As mentioned above, we decided to insert our plasmid constructs into E. coli expression cells to demonstrate the effect of IPTG induction on the expression of proteins from IPTG inducible promoters in constructs B and D.

We used two strains of E. coli BL21(DE3) which were not transformed with our constructs as negative controls. One of the negative controls and strains carrying plasmids with our constructs were induced at OD = 0.8 immediately after diluting overnight cultures to reach this OD. Fluorescence was measured 4 hours after induction with IPTG.

Samples from cultures of strains containing our constructs were also analyzed using fluorescence microscopy. Further details of the work with the fluorescence microscope can be found here: Wet lab, Characterization of construct B and D.

Analysis of GFP emission spectra of E.coli BL21(DE3)_pUCIDT_B

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Figure 30: GFP emission spectra measured in cultures of E.coli BL21(DE3) strains carrying construct B and negative controls.This graph shows the emission spectra of GFP in E.coli BL21(DE3) strains. Different samples analyzed in the experiment are labeled in the annotation of the graph. Two samples are negative controls (one of which was induced by IPTG at OD 0.8). The other two samples come from cultures of strains containing our construct B (one of which was induced by IPTG at OD 0.8). The scattered peaks in the left portion of the graph which do not correspond to the emission spectrum of GFP are the effects of fluorescence signal scattering on insoluble structures and overall heterogeneity of the measured sample. This scattering may occur during the work with cell suspensions. The green line indicates the emission maximum of GFP at 510 nm.

The presence of a typical GFP emission profile and the increase influorescence intensity compared to the negative controls confirm the presence of construct B in E.coli BL21(DE3) as well as the expression and correct function of GFP encoded by this construct.

There is however no significant difference between the fluorescenceintensity of BL21(DE3)_pUCIDT_B cultures with and without IPTG induction. This suggests that IPTG induction has no effect on the expression of target proteins.

Fluorescence microscopy of induced cultures:

Fluorescence microscopy of not-induced cultures:

Figure 31: Induced E. coli BL21(DE3) cells transformed with construct B under fluorescence microscope.Images of native unfixed samples of E. colicultures carrying construct B taken with a fluorescence microscope. This image shows BL21(DE3)_pUCIDT_B 2xIPTG which was induced with double concentration of IPTG. The image on the left is a brightfield image, the image in the middle was taken with a GFP filter and on the right is the overlay of the two previous images. Immersion oil was used. M = 52x

Figure 32: Uninduced E. coli BL21(DE3) cells transformed with construct B under fluorescence microscope.Images of native unfixed samples of E. colicultures carrying construct B taken with a fluorescence microscope. This image shows BL21(DE3)_pUCIDT_B 2xIPTG without IPTG induction. The image on the left is a brightfield image, the image in the middle was taken with a GFP filter and on the right is the overlay of the two previous images. M = 52x

Observations made using fluorescence microscopy correspond to the findingsfrom fluorescence spectra measurements and confirm the expression and correct folding of GFP in E. coli cultures transformed with construct B. GFP was detected in similar quantities in both induced and uninduced cultures of BL21(DE3)_pUCIDT_B . Induction does not affect the expression of our target protein.

The same experimental approach was used for BL21(DE3)_pUCIDT_D cells.

Analysis of GFP emission spectra of E.coli BL21(DE3)_pUCIDT_D

Figure 33: GFP emission spectra measured in cultures of E.coli BL21(DE3) strains carrying construct D and negative controls.This graph shows the emission spectra of GFP in E.coli BL21(DE3) strains. Different samples analyzed in the experiment are labeled in the annotation of the graph. Two samples are negative controls (one of which was induced by IPTG at OD 0.8). The other two samples come from cultures of strains containing our construct D (one of which was induced by IPTG at OD 0.8). The scattered peaks in the left portion of the graph which do not correspond to the emission spectrum of GFP are the effects of fluorescence signal scattering on insoluble structures and overall heterogeneity of the measured sample. This scattering may occur during the work with cell suspensions. The green line indicates the emission maximum of GFP at 510 nm.

Analysis of mScarlet-I emission spectra of E.coli BL21(DE3)_pUCIDT_D

Figure 34: mScarlet-I emission spectra measured in cultures of E.coliBL21(DE3) strains carrying construct D and negative controls.This graph shows the emission spectra of mScarlet-I in E.coli BL21(DE3) strains. Different samples analyzed in the experiment are labeled in the annotation of the graph. Two samples are negative controls (one of which was induced by IPTG at OD 0.8). The other two samples come from cultures of strains containing our construct D (one of which was induced by IPTG at OD 0.8). The scattered peaks in the left portion of the graph which do not correspond to the emission spectrum of mScarlet-I are the effects of fluorescence signal scattering on insoluble structures and overall heterogeneity of the measured sample. This scattering may occur during the work with cell suspensions. The green line indicates the emission maximum of mScarlet-I at 510 nm.

The presence of a typical GFP and mScarlet-I emission profiles and theincrease in fluorescence intensity compared to the controls confirmed the presence of construct D in E.coli BL21(DE3) as well as the expression and correct function of both fluorescent proteins encoded in this construct.

The issue observed when measuring GFP emission spectra of construct B ishowever also present here. There is no significant difference between the fluorescence intensity of BL21(DE3)_pUCIDT_D cultures with and without IPTG induction. This suggests that IPTG induction has no effect on the expression of target proteins.

These findings indicate that mScarlet-I and therefore also repressor cI areexpressed constitutively. Repressor cI should bind to our modified promotor P _Grac-OcI and inhibit the expression of genes downstream this promoter - namely sfGFP . Significantly lower intensity of sfGFP emission signal in samples of cultures of strains bearing construct D compared to mScarlet-I and sfGFP in samples with construct B might therefore suggest that our modified promoter can be repressed by the cI repressor although some leakage is present.

Further experiments would be helpful for confirming these conclusions. Wewere unfortunately unable to perform additional experiments due to the time constraints.

Fluorescence microscopy of uninduced cultures:

Figure 35: Uninduced E. coli BL21(DE3) cells transformed with construct D under fluorescence microscope.Images of native unfixed samples of E. coli cultures carrying construct D taken with a fluorescence microscope. This image shows BL21(DE3)_pUCIDT_D without IPTG induction. The image on the left is a brightfield image, the following image was taken with a GFP filter, the next image to the right was taken with mScarlet-I filter and on the right is the overlay of the three previous images. Immersion oil was used. M = 52x

Figure 36: Uninduced E. coli BL21(DE3) cells transformed with construct D under fluorescence microscope.Images of native unfixed samples of E. coli cultures carrying construct D taken with a fluorescence microscope. This image shows BL21(DE3)_pUCIDT_D without IPTG induction. The image on the left is a brightfield image, the following image was taken with a GFP filter, the next image to the right was taken with mScarlet-I filter and on the right is the overlay of the three previous pictures. Immersion oil was used. M = 88.2x

These images from fluorescence microscope confirm the expression andcorrect function of both GFP and mScarlet-I even in the absence of IPTG in BL21(DE3)_pUCIDT_D .

Observations made using fluorescence microscopy correspond to the findingsfrom fluorescence spectra measurements and confirm the expression and correct function of both GFP and mScarlet-I in E. coli cultures transformed with plasmid bearing construct D. Both GFP and mScarlet-I were detected in similar quantities in samples from induced and uninduced cultures of BL21(DE3)_pUCIDT_D . Induction does not have any effect on the expression of our target proteins.

b) Optimization of IPTG induction in E.coli strain BL21(DE3)

As we did not observe any difference in the expression of our target proteins in induced cultures compared to cultures which were not induced, we decided to try to optimize the IPTG concentration used for induction. The final concentration used in previous experiments was 100 μM IPTG. We decided to also two-fold higher concentration (200 μM IPTG). The emission spectra of samples from cultures induced with this increased concentration of IPTG were then analyzed using FluoroMax Plus.

We prepared two negative controls from cultures of strains which were not transformed with our constructs. One of the negative controls was induced with IPTG during inoculation. Samples BL21(DE3)_pUCIDT_B/D IPTG were induced during inoculation. Samples BL21(DE3)_pUCIDT_B/D 2xIPTG were firstly induced with 100 μM IPTG during inoculation and a second dose of IPTG was added 15 hours later. The emission spectra were measured 4 hours after the second dose of IPTG.

Analysis of GFP emission spectra of E.coli BL21(DE3)_pUCIDT_B:

Figure 37: Comparison of GFP emission spectra measured in cultures of E.coli BL21(DE3) strains carrying construct B induced with different concentrations of IPTG and negative controls.This graph shows the emission spectra of GFP in E.coli BL21(DE3). Different samples analyzed in the experiment are labeled in the annotation of the graph. Two samples are negative controls (one of which was induced by IPTG at inoculation). The other two samples are cultures containing construct B. Both of them were induced by IPTG already at the beginning of cultivation. An additional dose of IPTG was added to one of them after 15 hours. The scattered peaks in the left portion of the graph which do not correspond to the emission spectrum of GFP are the effects of fluorescence signal scattering on insoluble structures and overall heterogeneity of the measured sample. This scattering is expected when working with cell suspensions. The green line indicates the emission maximum of GFP.

Analysis of GFP emission spectra of E.coli BL21(DE3)_pUCIDT_D:

Figure 38: Comparison of GFP emission spectra measured in cultures of E.coli BL21(DE3) strains carrying construct D induced with different concentrations of IPTG and negative controls.This graph shows the emission spectra of GFP in E.coli BL21(DE3). Different samples analyzed in the experiment are labeled in the annotation of the graph. Two samples are negative controls (one of which was induced by IPTG at inoculation). The other two samples are cultures containing construct D. Both of them were induced by IPTG already at the beginning of cultivation. An additional dose of IPTG was added to one of them after 15 hours. The scattered peaks in the left portion of the graph which do not correspond to the emission spectrum of GFP are the effects of fluorescence signal scattering on insoluble structures and overall heterogeneity of the measured sample. This scattering is expected when working with cell suspensions. The green line indicates the emission maximum of GFP.

Analysis of mScarlet-I emission spectra of E.coli BL21(DE3)_pUCIDT_D:

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Figure 39: Comparison of mScarlet-I emission spectra measured in cultures of E.coli BL21(DE3) strains carrying construct D induced with different concentrations of IPTG and negative controls.This graph shows the emission spectra of mScarlet-I in E.coli BL21(DE3). Different samples analyzed in the experiment are labeled in the annotation of the graph. Two samples are negative controls (one of which was induced by IPTG at inoculation). The other two samples are cultures containing construct D. Both of them were induced by IPTG at inoculation. An additional dose of IPTG was added to one of them after 15 hours. The scattered peaks in the left portion of the graph which do not correspond to the emission spectrum of mScarlet-I are the effects of fluorescence signal scattering on insoluble structures and overall heterogeneity of the measured sample. This scattering is expected when working with cell suspensions. The red line indicates the emission maximum of mScarlet-I.

These measurements demonstrate that in both construct B and D, theexpression of both GFP and mScarlet-I is not influenced by the increase in IPTG concentration.

c) Measurement of fluorescence spectra in Bacillus subtilis

The emission spectra of our target fluorescent proteins were also measured in samples of cultures with B. subtilis strains bearing constructs B or D integrated into their chromosome. In the case of chromosomal integration, only one copy of the construct is present and therefore lower intensity of fluorescent signal then in E. coli , transformed with multicopy plasmids carrying our construct, can be expected. We used a positive control kindly provided by Dr. Krásný's research group - B. subtilis with GFP integrated in its chromosome downstream of a xylose-inducible promoter.

Unfortunately, we were unable to obtain positive B. subtilis control containing mScarlet-I integrated in chromosome.

B.subtilis_B and B.subtilis_D cultures were also analyzed using fluorescent microscopy.

Analysis of GFP emission spectra of B. subtilis_B:

Figure 40: GFP emission spectra measured in cultures of B. subtilis 168 strains carrying construct B and negative controls.This graph shows the emission spectra of GFP produced in B.subtilis. Different samples analyzed in the experiment are labeled in the annotation of the graph. The positive control has GFP gene integrated in its chromosome and was kindly provided by Dr. Kásný. The negative control is a strain of B.subtilis 168 without construct B. The last sample is a culture with construct B integrated into the chromosome. The scattered peaks in the left portion of the graph which do not correspond to the emission spectrum of GFP are the effects of fluorescence signal scattering on insoluble structures and overall heterogeneity of the measured sample. This scattering is expected when working with cell suspensions. The green line indicates the emission maximum of GFP.

Analysis of GFP emission spectra of B. subtilis_D:

Figure 41: GFP emission spectra measured in cultures of B. subtilis 168 strains carrying construct D and negative controls.This graph shows the emission spectra of GFP in B.subtilis. Different samples analyzed in the experiment are labeled in the annotation of the graph. The positive control has GFP gene integrated in its chromosome and was kindly provided by Dr. Kásný. The negative control is a strain of B.subtilis 168 without construct D. The last sample is a culture with construct D integrated into the chromosome. The scattered peaks in the left portion of the graph which do not correspond to the emission spectrum of GFP are the effects of fluorescence signal scattering on insoluble structures and overall heterogeneity of the measured sample. This scattering is expected when working with cell suspensions. The green line indicates the emission maximum of GFP.

Analysis of mScarlet-I emission spectra of B. subtilis_D:

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Figure 42: mScarlet-I emission spectra measured in cultures of B. subtilis168 strains carrying construct D and negative controls.This graph shows the emission spectra of mScarlet-I in B.subtilis. Different samples analyzed in the experiment are labeled in the annotation of the graph. The negative control is a strain of B.subtilis 168 without construct D. The last sample is a culture with construct D integrated into the chromosome. The scattered peaks in the left portion of the graph which do not correspond to the emission spectrum of mScarlet-I are the effects of fluorescence signal scattering on insoluble structures and overall heterogeneity of the measured sample. This scattering is expected when working with cell suspensions. The red line indicates the emission maximum of mScarlet-I.

Fluorescence microscopy of B. subtilis_B and B. subtilis_D cultures:

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Figure 43: Negative control - B. subtilis 168 cells with no insert in its chromosome - under fluorescence microscope.Images of native unfixed samples of B. subtilis taken with a fluorescence microscope. This image shows a culture of B. subtilis 168 with no insertion in it's chromosome. The image on the left is a brightfield image, the image in the middle was taken with a GFP filter and on the right is the overlay of the two. Immersion oil was used. M = 52x

Figure 44: B. subtilis 168 cells transformed with construct B under fluorescence microscope.Images of native unfixed samples of B. subtilis taken with a fluorescence microscope. This image shows B. subtilis_B . The image on the left is a brightfield image, the image in the middle was taken with a GFP filter and on the right is the overlay of the two. Immersion oil was used. M = 52x

Figure 45: B. subtilis 168 cells transformed with construct D under fluorescence microscope.Images of native unfixed samples of B. subtilis taken with a fluorescence microscope. This image shows B. subtilis_D . The image on the left is a brightfield image, the following image was taken with a GFP filter, the next image to the right was taken with a mScarlet-I filter and on the right is the overlay of the three. Immersion oil was used. M = 52x

Analysis of GFP emission spectra of B. subtilis_Busing FluoroMax Plus confirmed the expression and correct function of sfGFP . The emission profile is comparable to the positive control provided by Dr. Krásný, which also has a GFP gene inserted into its chromosome.

Analysis of emission spectra of both of our target fluorescent proteins in B. subtilis_D using FluoroMax Plus show the absence of a typical emission profile or significant increase in fluorescence intensity for both GFP and mScarlet-I compared to the negative control. This indicates that there are no or very little fluorescent proteins produced by B. subtilis_D cells.

Observations made using fluorescence microscopy however indicate a presenceof small fluorescent signals of GFP in B. subtilis_B and GFP and mScarlet-I in B. subtilis_D . There was no fluorescence detected in the negative control, so the signals from B. subtilis_B and B. subtilis_D should be the result of integration of our constructs in bacterial chromosome.

To confirm expression of sfGFP and mScarlet-I genes integrated in the B. subtilis_D chromosome, the spectrum analysis would need to be optimized.

Conclusion

To conclude everything, we would like to briefly summarize the successes achieved in the wet lab. We have managed to transform E. coli strains JM109 and BL21(DE3) with three out of four constructs (namely constructs B, C and D) and we achieved the same success with B. subtilis . We have verified the cloning and transformation of our constructs using restriction digestion, PCR and sequencing. We were able to demonstrate the expression of fluorescence proteins from constructs B and D, both of which proved to be functional under the fluorescence microscope in B. subtilis . Last but not least, we were able to figure out how to bipas the issues with the synthesis of our construct A by using Golden Gate assembly (which is not included in the Result section - due to lack of time to perform this experiment until wiki freeze).

What could be improved?

Because we know failures are part of every successful experiment, and our project was no exception, we want to talk about them and share the lessons we learned from our mistakes. In order to correct one's failings, one must first recognise them. When subcloning our constructs from the original pUCIDT plasmids into the pDG destination vectors, we naively choose the same selection marker for both vectors (resistance to ampicillin). This flaw in our experimental design made successful subcloning into the shuttle vector very difficult, especially in the case of construct C, as we were unable to distinguish colonies containing successfully ligated pDG vectors with our construct from colonies contaminated with pUCIDT.

Another important lesson was learned at the end of our experimental work. After verifying the expression of functional fluorescence proteins from our constructs, we still need to perform more experiments as well optimize some which did not work as expected to prove the function of our system as a whole. Unfortunately, we did not manage to do this due to time constraints. For future reference, we will know that a longer period of time should be set aside for measurements and optimization of characterization of each construct.