We faced the challenge of developing a project ranging from characterization of non-standard growth media, through BC cultivation, hardware engineering, computer simulation, strain design, plasmid engineering, transformations, and applications of our final product (BC). Due to the broadness of the project and the limited time for its execution, we developed multiple fronts in parallel. Here we present some of the highlights of our work.
A consistent transformation protocol is crucial for any strain engineering project. Thus, we invested much work in establishing the ideal conditions for K. rhaeticus AF1 electroporation. Initially, we based our attempts on the strategies used for the transformation of K. rhaeticus iGEM (Goosens, Vivianne J., et al., 2021), however, all of our experiments failed. . Other literature sources were also used in an attempt to carry out the transformation, but none of them resulted in success.
After troubleshooting parameters such as preparation of competent cells, plasmid selection markers, plasmid replication origins, the intensity of electroporation, cuvette brands, cell density, and many other parameters, we decided to change the suggested antibiotic concentration on our HS agar plates. After all of our frustrating attempts, we finally established a consistent protocol for K. rhaeticus AF1 transformation.
Optimized Komagataeibacter rhaeticus AF1 transformation protocol by electroporation (protocol)
Komagataeibacter is a non-conventional chassis in which traditional E. coli replication origins are not functional. Hence, plasmid replication requires broad-spectrum origins such as RK2, pBBR1 or RSF1010. Within iGEM´s 2022 distribution kit there were 6 plasmids with such requirements, namely the GoldenGate level 1: BBa_J428346, BBa_J428347, and BBa_J428349 (KanR), and the GoldenGate level 2: BBa_J428366, BBa_J428367, and BBa_J428369 (SpecR). This posed a potential problem to our design strategy as, to the best of our knowledge, there is no report of successful use of the SpecR marker in K. rhaeticus. Thus, we design a strategy to replace the KanR and SpecR marker from level 1 and 2 plasmids (respectively) with both AmpR and CmR. This was accomplished by designing the primers JUMP_mF and JUMP_mR to amplify BBa_J428346, BBa_J428347, BBa_J428349, BBa_J428366, BBa_J428367 and BBa_J428369 excluding the KanR and SpecR selection markers (for details, see parts section). In parallel, we designed primers AmpR_F and AmpR_R to amplify the Ampicillin resistance cassette from BBa_J428385 and primers CmR_F and CmR_R to amplify the chloramphenicol resistance cassette from BBa_J428357. Employing digestion with SapI and ligation with T4 DNA ligase, we succeeded in assembling new level 1 and level 2 Komagateibacter compatible plasmids with different marker options.
Thus, we succeeded in constructing the following plasmids:
| New backbones | Details | Internal Code | Validation |
|---|---|---|---|
| BBa_K4435301 | amplification of BBa_J428346 with JUMP_mF and JUMP_mF; amplification of antibiotic resistance marker; Golden gate cloning with SapI | 2K to AmpR | phenotype |
| BBa_K4435302 | amplification of BBa_J428347 with JUMP_mF and JUMP_mF; amplification of antibiotic resistance marker; Golden gate cloning with SapI | 2M to AmpR | phenotype |
| BBa_K4435303 | amplification of BBa_J428349 with JUMP_mF and JUMP_mF; amplification of antibiotic resistance marker; Golden gate cloning with SapI | 4A to AmpR | |
| BBa_K4435304 | amplification of BBa_J428366 with JUMP_mF and JUMP_mF; amplification of antibiotic resistance marker; Golden gate cloning with SapI | 6K to AmpR | phenotype |
| BBa_K4435305 | amplification of BBa_J428367 with JUMP_mF and JUMP_mF; amplification of antibiotic resistance marker; Golden gate cloning with SapI | 6M to AmpR | phenotype |
| BBa_K4435308 | amplification of BBa_J428346 with JUMP_mF and JUMP_mF; amplification of antibiotic resistance marker; Golden gate cloning with SapI | 2K to CmR | |
| BBa_K4435309 | amplification of BBa_J428347 with JUMP_mF and JUMP_mF; amplification of antibiotic resistance marker; Golden gate cloning with SapI | 2M to CmR | phenotype |
| BBa_K4435310 | amplification of BBa_J428349 with JUMP_mF and JUMP_mF; amplification of antibiotic resistance marker; Golden gate cloning with SapI | 4A to CmR | |
| BBa_K4435311 | amplification of BBa_J428366 with JUMP_mF and JUMP_mF; amplification of antibiotic resistance marker; Golden gate cloning with SapI | 6K to CmR | phenotype |
| BBa_K4435312 | amplification of BBa_J428367 with JUMP_mF and JUMP_mF; amplification of antibiotic resistance marker; Golden gate cloning with SapI | 6M to CmR | |
| BBa_K4435315 | add ODD 4 TUs adapter, derived from BBa_J428366: pJUMP42x-2A SpecR Type IIS Level 2 vector. Origin RK2 (broad-host-range); | 6K_red | phenotype |
| BBa_K4435316 | add ODD 4 TUs adapter, derived from BBa_J428367: pJUMP43-2A SpecR Type IIS Level 2 vector. Origin pBBR1 (medium-copy, broad-host-range) | 6M_red | phenotype |
| BBa_K4435317 | add ODD 4 TUs adapter, derived from BBa_J428369: pJUMP45-2A SpecR Type IIS Level 2 vector. Origin RSF1010 (Broad-host-range) | 8A_red | phenotype |
| BBa_K4435306 | add ODD 4 TUs adapter to BBa_K4435304 | 6K to AmpR_red | phenotype |
| BBa_K4435307 | add ODD 4 TUs adapter to BBa_K4435305 | 6M to AmpR_red | phenotype |
| BBa_K4435313 | add ODD 4 TUs adapter to BBa_K4435311 | 6K to CmR_red | phenotype |
| BBa_K4435314 | add ODD 4 TUs adapter to BBa_K4435312 | 6M to CmR_red |
Phenotipic validation: Image illustrating E. coli strains harbouring novel plasmids and streaked onto plates with the indicated antibiotics.
Our final proof of success is the transformation of Komagataeibacter with part BBa_K4435305, which has the new antibiotic resistance cassette and shows it’s efficiency in Komagataeibacter rhaeticus AF1 (5 and 10 mg/L ampicillin allowed background growth of cells electroporated but without plasmids; 400 mg/L - the recommended ampicillin concentration in the literature - exceeded the tolerable levels for the AF1 strain; 25 mg/L of ampicillin did not allow the growth of cells without plasmids but selected positive transformants). All the new plasmids with swapped markers are await for sequencing.
Our engineering strategy for the replacement of bcsZ or bcsA promotors requires the genomic integration of cassettes with include: 1 kb of homology to the region 5´the integration locus, recyclable antibiotic resistance marker (AmpR flanked by FRT recombination sites), transcriptional unit encoding the LexRO transcriptional repressor under the control of strong to medium promotors, and a final module encoding the LexRO binding region (operator) followed by RBS and 1 kb of homology to the region 3´the integration locus. This design requires level 2 assembly of 4 transcriptional units (basic TUs assembled in pOdd1 to pOdd4 plasmids).
In parallel, we also cloned in level 0 plasmids genes (from K. rhaeticus and K. medellinensis) encoding key proteins in the pathway that converts glucose to BC (awaiting sequencing results). These will be assembled into transcriptional units TU1 to TU4 in pOdd1 to pOdd4 plasmids. Concomitant overexpression of 4 different proteins requires their assembly into level 2 Komagataeibacter compatible plasmids.
Unfortunately, the available backbones did not meet our needs, which requires the presence of SapI restriction with ends complementary to 5´of TU1 (pOdd1) and 3´of TU4 (pOdd4), thus we designed primers AdapterTU_reD_F and AdapterTU_reD_R and amplified the red reporter from BBa_J04452. PCR products were assembled by GoldenGate using BsaI and T4 DNA ligase with level 2 plasmids. Successful assembly yielded red colonies and original unedited plasmids yielded green colonies.
New backbones (Type IIs compatible):
| New backbones | Source | Internal Code | Validation |
|---|---|---|---|
| BBa_K4435315 | add ODD 4 TUs adapter, derived from BBa_J428366: pJUMP42x-2A SpecR Type IIS Level 2 vector. Origin RK2 (broad-host-range) | 6K_red | phenotype |
| BBa_K4435316 | add ODD 4 TUs adapter, derived from BBa_J428367: pJUMP43-2A SpecR Type IIS Level 2 vector. Origin pBBR1 (medium-copy, broad-host-range) | 6M_red | phenotype |
| BBa_K4435317 | add ODD 4 TUs adapter, derived from BBa_J428369: pJUMP45-2A SpecR Type IIS Level 2 vector. Origin RSF1010 (Broad-host-range) | 8A_red | phenotype |
| BBa_K4435306 | add ODD 4 TUs adapter to BBa_K4435304 | 6K to AmpR_red | phenotype |
| BBa_K4435307 | add ODD 4 TUs adapter to BBa_K4435305 | 6M to AmpR_red | phenotype |
| BBa_K4435313 | add ODD 4 TUs adapter to BBa_K4435311 | 6K to CmR_red | under construction |
| BBa_K4435314 | add ODD 4 TUs adapter to BBa_K4435312 | 6M to CmR_red | phenotype |
Considering the BC synthesis pathway we were able to identify key enzymes involved in cellulose production, specially when Glucose is the main carbon source for this biosynthesis. Trough our modeling, we confirm which main enzymes could be overexpressed and help in the production of cellulose.
After glucose (GLC) import we see this conversion into D-glucose-6-phosphate (G6P) by glucokinase (GK), which is then converted into D-glucose-1-phosphate (G1P) by UTP-glucose-1-phosphate uridylyltransferase (galU). From that UDP-glucose (UDPG) is produced by hosphoglucomutase (pgm), which requires UTP by nucleoside diphosphate pyrophosphorylase (ndp) production. UDPG works as a substrate for cellulose synthesis by bcsA, which requires cyclic diguanylic acid (C-di-GMP) produced by diguanylate cyclase (dgc).
In order to obtain experimental results consistents with the modelling prediction, identifying the limiting enzyme in the pathway, we amplified genes encoding GK, galU, pgm and ndp from genomic DNA of K. rhaeticus AF1 and K. medellinensis, along with the synthesis of dgcI. These are being cloned into BBa_J428381, BBa_J428382, BBa_J428383, and BBa_J428384, under the control of the strong constitutive promotor BBa_K4435012, RBS BBa_K4435015 and terminator BBa_K4435017 (given us level 1 composite parts BBa_K4435120 to BBa_K4435127).
The constructs were transformed into E. coli, which grew efficiently, proving the antibiotic resistance and, consequently, the right assembly of the plasmids. Some of it’s resistant colonies were isolated, plasmid prepared, and analyzed by agarose gel electrophoresis. The band sizes show many candidates were sent for sequencing. Further colonies are being selected for constructs where we have not selected plasmids of the correct size in our first attempt (only 1 colony was selected per plate in our first screen). We are confident that we have level 0 plasmids for all our basic parts (awaits confirmation).
Parts listed below will be assembled into transcriptional units (TU1 to TU4) in pOdd 1 to 4 plasmids for further level 2 assembly into BBa_K4435306 or BBa_K4435307. These can then be transformed into K. rhaeticus and BC production yields are quantified.
Level 0 (Type IIs compatible):
| Level 0 Part | Short name | Origin and protein cloned |
|---|---|---|
| BBa_K4435004 | Kr_galU_TU1 | K. rhaeticus AF1 galU_UTP-glucose-1-phosphate uridylyltransferase |
| BBa_K4435005 | Kr_pgm_TU2 | K. rhaeticus AF1 pgm_Phosphoglucomutase |
| BBa_K4435006 | Kr_ndp_TU3 | K. rhaeticus AF1 ndp_Nucleoside diphosphate pyrophosphorylase |
| BBa_K4435007 | Kr_GK_TU4 | K. rhaeticus AF1 GK_Glucokinase |
| BBa_K4435008 | Km_galU_TU1 | K. medellinensis galU_UTP-glucose-1-phosphate uridylyltransferase |
| BBa_K4435009 | Km_pgm_TU2 | K. medellinensis pgm_Phosphoglucomutase |
| BBa_K4435010 | Km_ndp_TU3 | K. medellinensis ndp_Nucleoside diphosphate pyrophosphorylase |
| BBa_K4435011 | Km_GK_TU4 | K. medellinensis GK_Glucokinase |
| BBa_K4435003 | dgc1_TU4 | dgc1_diguanylate cyclase |
Aiming to improve our cellulose production and detach dark (no BC production) and light (bcs induction) phases of the process, we designed and built a two step bioreactor. As our project proposes, Komagataeibacter rhaeticus AF1 should have a period of growth, in the absence of light, followed by activation of it’s bcs operon by luminous stimulus. To predict the best moment to start the induction of cellulose production, resulting in a higher amount of final product, we comply our math model. The conditions of presence or privation of light and even the change-over them can be respected in the developed hardware. Experimental tests demonstrate the the efficiency of bioreactor by the exponencial growth of E. coli and suggested a big condition advantage for obligate aerobic organisms.
The operation of the bioreactor is simple, it works with (a) the culture medium with genetically modified KAF1 where as soon as it reaches a maximum OD, without harming the colony, it will be inserted into our closed system (b) through a hose that connects (a) and (b). And thus we activate the blue light that is contained in its lid. In this way we can fully control the cellulose production. All materials in the bioreactor are sterile.
Komagataeibacter was grown in liquid HS media, which has D-glucose as its sugar source. We asked ourselves if the bacteria use this glucose as a substrate for cellulose production. So, in order to evaluate glucose consumption, we used a quantification technique based on High Performance Liquid Chromatography (HPLC).
For this analysis, we injected 5 samples collected from different days of bacterial growth, to compare their sugar elution profile to a standard sample of monosaccharides and short-chain carbohydrates. This way it was possible to identify and quantify the sugars present in our sample by integrating their corresponding peaks.
The media samples’ elution profiles showed peaks that matched with glucose and, the short-chain sugar made of three units of glucose, cellotriose. It is possible to see glucose’s concentration decrease, as well as the cellotriose’s concentration increase as the growth days go by in the chromatograms. This observation shows that the glucose monomer is consumed by the bacteria and glucose polymers are being formed, as glucose decrease coincides with cellotriose’s increase (Figure 1).
The chromatograms obtained are available here
We also wondered about glucose concentration in the alternative agroindustrial residue media, in a way to predict bacteria growth. So we measured it in the HPLC as well. The chromatogram in figure shows a concentration of 3.1836 g/L of glucose and the presence of other carbohydrates that were not identified, as they are not in the standard sample of monosaccharides and short-chain carbohydrates.
With the goal of producing BC membranes that could coat complex 3-dimensional structures, we investigated the requirements for unfolding the surface of different objects onto 2D. By using the perimeter of this 2D object as a guide, we devised a strategy to 3D print molds for the production of BC sheets. As proof of principle, we constructed molds representing the iGEM logo and the surface of a sphere and used to produce BC sheets.
An exceptional application of BC is its use as a scaffold for cell culture, once it forms colloidal level dispersions in an aqueous medium and shows strong and crystalline structure, boosting cell culturing experiments.(Madhushree Bhattachary et al., 2012). In previous studies, bacterial cellulose has already been tested and shown to be convenient as a scaffold for hard tissues (eg. bone and cartilage). Aiming to evaluate the adherence of distinct cell lines in BC and their efficiency as scaffolds we perform a vast number of assays during our project. First of all, we used the lineage of human skin fibroblast NIH3T3 and reached a praiseworthy cell adhesion in all the groups. In the figure below we can see cells under brightfield microscopy, with a morphology slightly different from the expected pattern, but adhered to the blanket as expected.
The second batch of tests was with the SK-MEL-28 strain of human melanoma, in which we obtained good adhesion to bacterial cellulose, however, morphologically, they do not appear to undergo cell differentiation, since they have a phenotype similar to that found when the cell does not is adhered to the substrate. In addition to these points, we could also observe that cell adhesion is greater in thinner cellulose when compared to thicker cellulose.
In view of the results obtained, we were able to identify some points that are amenable to the improvement of our design of experiment, as a way of standardizing the blankets used for cultivation, minimizing the effects of variable parameters. In this scenario, we rethink our design and proposed the use of a 3D mold (hardware) that allows the standardization of BC production in optimal size and thickness for cell culture. After the growth of these new cellulose sheets, we ran an assay with C2c12 mouse myoblasts, which shows the efficiency of cell adherence and differentiation rate. The standardization of the sheets seems to be an effective improvement for cell culture as we can see from the pictures. Some observations allowed during these experiments propose the requirement of thinner sheets.
