Week 0

Prior to the lab

In the month prior to starting in the lab, our wet lab instructor Gabriel introduced us to the Golden Gate Cloning system. During this time, we designed plasmids for the different levels of the cloning process, ordered our gene fragments and supplies for the lab, and planned our experiments accordingly. Our instructor, Zaira, guided us through the process of designing an sgRNA for the knockdown of the PDE gene. This preparation laid the foundation for the upcoming lab work.

Week 1

8.7 – 12.7

Golden Gate Level 0

We made competent cells from E. coli SY327 and transformed level 0 parts from the iGEM kit. Heat shock transformation was also performed with the pBP plasmid, which served as the level 0 backbone for our ordered gene fragments. Successful transformations were stored in glycerol, while unsuccessful assemblies were repeated.

CRISPRi

We miniprepped E. coli pSCB2 to extract the pSCB2 plasmid, which was then used to insert our sgRNA via CRISPRi inverse PCR. After confirming the amplification by gel electrophoresis, we treated the samples with DpnI to remove the native methylated DNA. This ensured that only our unmethylated plasmid containing the sgRNA was present in the sample. Following ligation, the pSCB2-sgRNA plasmid was transformed into E. coli SY327.

Week 3

22.7 – 26.7

Golden Gate Level 0 & Level 1

The failed level 0s were reassembled, though some were still unsuccessful. Successful level 0s were miniprepped and used to assemble our level 1 testing and final construct plasmids. White colonies from the level 1 testing constructs were sent for sequencing directly (as our level 1 backbones contain GFP as a reporter gene), while the final constructs were incubated over the weekend.

Biofilm Staining

We performed a colony PCR with colonies from the triparental conjugation, which showed no difference to the control (which lacked the helper strain), suggesting the helper strain may not be necessary. We prepared overnight cultures for the polysaccharide staining assay, incubating our P. sp. IsoF dCas9 pSCB2-sgRNA as well as the negative control (IsoF dCas9) both with and without rhamnose. The rhamnose-treated cultures were expected to show increased polysaccharide production, which could be seen by eye already the next day. The overnight cultures were then plated on Congo red derivative plates and incubated over the weekend.

Week 5

5.8 – 9.8

Golden Gate Level 1

After further research we decided to redo the level 1 plasmids using higher concentrations of gene fragments and backbones and adding a buffer specific to our assembly enzyme, BsaI.

c-di-GMP Assay

We conducted a preliminary c-di-GMP assay to optimize the conditions for measuring c-di-GMP production, as the results of last week’s assay were unsuccessful. Cells were either unwashed or washed with saline solution and adjusted at different values of OD600. The pSCB2-sgRNA plasmid was used as a negative control, while a strain with an upregulated DGC was used as a positive control. Despite these adjustments the assay still did not reflect the expected difference in c-di-GMP production.

Week 6

12.8 – 16.8

Golden Gate Level 1

We repeated the level 1 plasmid assembly, doubling the concentrations of the backbone and gene fragments. Despite this, the number of white colonies did not increase, indicating that the efficiency of assembly did not improve. Sequencing of the white colonies revealed that the cloning was unsuccessful.

C-di-GMP Assay

Following our instructor's advice, we repeated the preliminary measurement, adjusting to lower values at OD600 and used saline solution instead of LB to reduce background noise. We focused on controls to optimize conditions instead of testing our DGC constructs. Negative controls included an upregulated PDE in P. sp. IsoF (pBBR1MCS5 PA5295) and an empty plasmid (pBBR1MCS5) in P. sp. IsoF, while an upregulated DGC in P. sp. IsoF served as a positive control (pBBR1MCS5 yedQ). The results reflected the expected c-di-GMP levels, particularly at low OD600 values.

Testing our DGCs in E. coli under the optimized conditions showed inconclusive results. We believe this is because the DGCs are not in their native strain, leading to the absence of other essential metabolic steps required for c-di-GMP upregulation. We decided to conjugate our DGCs into the P. sp. IsoF strain.

Toxin Testing

We conducted a preliminary toxin assay using rhamnose induced overnight cultures. We measured OD600 over a 16-hour period expecting that the toxin-induced cultures would exhibit reduced growth. However, the rhamnose-treated cultures displayed a higher OD600, leading us to question whether the OD is a good indicator of cell death, whether the cells were using rhamnose as an extra source of energy and whether our rhamnose promoter might be the issue.

Week 7

19.8 – 23.8

Adapting Plasmid Backbones

We attempted triparental conjugation to transfer our different testing DGC constructs (pT21Rhyz01 to pT21Rhyz05) from E. coli SY327 to P. sp. IsoF dCas9, but the conjugation was unsuccessful. Upon further research, we discovered that our plasmid is not replicative in P. sp. IsoF. Instead of recloning them into a replicative plasmid using Golden Gate assembly, we opted for digestion-ligation into the pBBRMCS5 plasmid, which is commonly used for gene expression in P. sp. IsoF. We amplified the transcription units on the above-mentioned plasmids with recognition sites for specific restriction enzymes (both in the MCS of the pBBRMCS5 plasmid) and then performed digestion-ligation into pBBRMCS5. The first attempt was unsuccessful, probably due to insufficient digestion, so we repeated it the next week.

Since none of our previously used backbones are replicative in P. sp. IsoF we had to adapt our plasmids. We identified three backbones in the iGEM distribution kit that are replicative in P. sp. IsoF, namely pJUM23-1A, pJUMP43-2A and pJUMP45-2A and transformed them into E. coli SY327.

Toxin Testing

We decided to test the functionality of our rhamnose promoter, to ensure that this is not the problem with our toxin. We did so using a plasmid that included RFP as a reporter gene. We performed a preliminary test which, after an overnight incubation with rhamnose, exhibited a red color, confirming that the rhamnose promoter was active. To quantify the promoter’s activity at different rhamnose concentrations and the potentially toxic effect of rhamnose on the cell, we performed an overnight growth assay, measuring OD600 and fluorescence. This showed that the promoter’s maximum activity with the minimum negative effects of rhamnose was at 1%.

Week 8

26.8 – 30.8

Golden Gate Level 1

After encountering issues with our initial Golden Gate cloning attempts, we had to reclone many of our level 1 plasmids. We used the pJUMP23-1A backbone, as this one is replicative in P. sp. IsoF. We ordered our transcription units as a single unit to improve cloning efficiency. After talking to our Wet Lab advisor, we adjusted our protocol to a 3:1 ratio of gene fragment to backbone (instead of a 1:1 ratio) and transformed less of the Golden Gate assembly. Most of the colonies were white, indicating the Golden Gate assembly had a much higher efficiency than the previous attempts. Sequencing the colonies confirmed that 17 out of 20 plasmids were cloned successfully.

Toxin Testing

Given that the three toxin testing plasmids (pT41Rhyz01, pT41Rhyz02, pT41Rhyz03), shared the same non-replicative backbone as the DGC plasmids, we applied the same amplification and digestion-ligation strategy with pBBRMCS5. However, this process failed and was repeated the following week. Our toxin test failed to show the expected effect in both E. coli SY327 and E. coli DH5alpha, due to a gyrase mutation conferring resistance to CcdB. After further research we identified E. coli HB101 as suitable and transformed our plasmids into this strain.

Digestion-Ligation of DGC Testing Plasmids

Since the digestion-ligation of the DGC Transcription Units failed last week, we repeated the experiment. Extending the digestion period allowed us to successfully clone the testing plasmids with the five different DGCs into the pBBRMCS5 plasmid.

Week 9

2.9 – 6.9

Biofilm Staining & c-di-GMP Assay

This week we conducted further testing on different DGC constructs, including two of our selfmade DGC mutations. We first conjugated our plasmids into P. sp. IsoF via triparental conjugation. We then set up two experiments to measure the c-di-GMP expression of our different DGCs (WspR wt, a WspR mutation, DGC IsoF wt and two DGC IsoF mutations), our knocked down PDE as well as our controls (P. sp. IsoF dCas9, P. sp. IsoF with an empty plasmid, the upregulated DGC yedQ).

We first measured the c-di-GMP levels through fluorescence intensity. For this we inoculated the colonies with rhamnose once overnight and once for 5 hours only, which produced more promising results than the overnights. For our second experiment, we stained the biofilms by plating these strains on plates with a Congo red-derived dye and inoculating them over the weekend. We analyzed the results of the staining the following week.

Golden Gate Cloning

Following previous challenges with Golden Gate, we repeated the cloning of level one and two plasmids. These included testing plasmids for sensing, toxins and a strain with our mutated DGC to inoculate our plants with our final construct plasmids. According to the cPCR gel of the level 2 cloning, it failed. After troubleshooting, we decided to redo the cPCR the following week using Q5 Polymerase instead of GoTaq, as the Q5 is more suited for amplification of sequences as long as our level 2 plasmids.

Sensing

In preparation for the sensing experiment next week, we did a triparental conjugation of our plasmid into P. sp. IsoF.

Toxin Tests

After difficulties with our toxin tests in the previous weeks, we chose E. coli HB101 as a new ccdB sensitive strain to test our toxin on. We performed a triparental conjugation of our plasmid into E. coli HB101. Unfortunately, the cells were not affected by the toxin. After some trouble shooting we decided to try the toxin test in two further strains, E. coli MC1061 and E. coli CSH50.

Week 10

9.9 – 13.9

Golden Gate Cloning

The colony PCR of last week’s Golden Gate assembly indicated that the cloning didn’t work. To ensure the cPCR wasn't the issue we repeated it with more colonies, again using Q5 polymerase instead of GoTaq for better long-sequence amplification. This second cPCR also came back negative. After troubleshooting, we decided to increase the insert to vector ratio to 5:1 and repeated the assembly of the remaining level 1 and level 2 plasmids.

Biofilm Staining

A second biofilm staining assay of the strains containing the different DGCs and the sgRNA to knockdown the PDE was conducted, since there was a mistake in the controls of the last assay. This time we included a negative control: a strain containing an upregulated PDE.

Toxin Testing

Both E. coli CSH50 and E. coli MC1061 showed no significant response to the toxin, as the cells continued growing. For the sake of being thorough and due to time constraints, we conjugated and tested our toxin constructs in P. sp. IsoF once more with no significant effect.

Sensing

Colony PCR confirmed that the cloning of our sensing plasmid was successful. After the first assay failed, further research revealed that the xylose-XutR complex binds not to the Pxut itself, but to an operator sequence located after it, which was missing in our construct. To address this, we planned to add the missing sequence via digestion-ligation or PCR-amplification and blunt-end ligation, and designed primers accordingly.

Gibson Assembly

We used Gibson assembly to replace spectinomycin resistance with kanamycin resistance on a backbone, as both backbones initially had the same resistance, and this change was necessary to select for both of our final plasmids in the same bacterium. After amplifying the level 2 backbone and kanamycin resistance from a level 1 backbone with specific primers, we ran a gradient PCR to improve efficiency and assembled them into a new level 2 backbone. We sent one promising colony for sequencing and will get the results next week.

Plant Experiments

We wanted to test the effect of our c-di-GMP regulating constructs on the growth of plants in wet and dry conditions. Unfortunately, as the cloning of our own DGC system with a constitutive promoter didn’t work in time, we used P. sp. IsoF pBBR1MCS5 yedQ as a comparable construct to represent how a DGC overexpression can still have an effect on plant growth. Therefore, we inoculated MicroTom plants (Solanum lycopersicum) with P. sp. IsoF pBBR1MCS5 yedQ, a PDE overexpression (P. sp. IsoF pBBR PDE), our strain with the empty plasmid (P. sp. IsoF wt pBBR1MCS5) and LB as a negative control. We collected data from 4 plants as a T0 measurement.

Week 11

16.9 – 20.9

Golden Gate Level 2

We conducted a Golden Gate assembly of our feedback loop plasmids (pT32Rhyz01-pT32Rhyz03) and our final construct plasmids (p21Rhyz04 and p22Rhyz04), which contained all our target constructs. To prevent self-ligation of the backbone, we pre-digested it and treated it with FastAP phosphatase. However, the colony PCR results were inconclusive, after sending them for sequencing.

c-di-GMP Assay

We repeated the c-di-GMP assay using the same strains as in previous experiments. Our P. sp. IsoF DGC mutant, R196A, showed the highest levels of c-di-GMP.

Biofilm Staining

We examined the biofilm staining plates under a microscope and captured images for analysis. Results indicated that the WspR mutation exhibited the highest biofilm formation, while the controls (P. sp. IsoF pBBR1MCS5 empty plasmid and P. sp. IsoF wt) showed the least.

Sensing

We performed the PCR amplification and blunt end ligation to introduce the operator sequence into our sensing plasmid. After transformation into E. coli SY327 we sent some colonies for sequencing, which unfortunately came back as incorrect as the primer bound to the RBS on the second transcription unit.

Plant Experiments

We did a colony-forming unit (CFU) count on the dilution series plates with countable colonies (between 30-300). Additionally, we measured the dry weight of the plants that had been drying in the oven over the weekend. The CFU count showed some unexpected inconsistency between the different samples, indicating that future results should be interpreted with caution. The dry weight of the aerial part of the plants matched our expectation from the previous wet weight measurements.

Week 12

23.9 – 27.9

C-di-GMP Assay

We conducted our final c-di-GMP assay, once again using various strains to compare the c-di-GMP levels across our DGC and PDE constructs. Consistent with previous results, the DGC R196A variant exhibited the higher c-di-GMP levels than the wildtype sequence.

Toxin Testing

We modified the toxin test by plating adjusted overnights containing our toxin construct, induced by the rhamnose promoter, onto rhamnose plates with varying concentrations (0%, 1%, and 1.5%). This experiment was conducted for both E. coli and P. sp. IsoF, using IsoF with an empty plasmid (pBBR1MCS5) and E. coli with a plasmid containing GFP as controls.

Sensing

As the sequencing of our sensing plasmid, in which we tried to implement the operator sequence with PCR amplification and blunt-end ligation returned as incorrect, we went forward with the oligo annealing as a second option to introduce the missing operator sequence. After the annealing reaction, ligation and transformation we sent for sequencing, the results of which we got the following week.

Swimming Motility Assay

We assessed the motility of bacteria carrying our different DGC constructs, using the same strains from our c-di-GMP assays, as reduced motility supports biofilm formation. As anticipated, the DGC constructs exhibited lower motility compared to the control strains. The motility assay was conducted twice.

Plant Experiments

We collected data from the remaining plants inoculated in Week 10, which involved measuring the aerial mass and plating a dilution series of bacteria from the rhizosphere. The dry weight of the plants was recorded the following week.