Goal:

Design NOS construction and express Nitric Oxide Synthase (NOS).

Achievements:

1. Construction of NOS

2. Run SDS-PAGE to confirm expression in E. coli

This year, our team aims to reduce intraocular pressure (IOP) through the contact lens with engineered E. coli that can produce Nitric Oxide (NO). We aimed to make our plasmid express NOS and support our bacteria to bind to the contact lenses. Since E. coli doesn't produce nitric oxide, we ordered a sequence containing a lacO-T7 promoter, B0034 RBS, and bsNOS. Then, we ligate the sequence by inserting it into the PUC plasmid and combining it with another sequence (pLac, RBS, csgA, RBS, csgD, LacI) from IDT and transformed into E. coli DH5-Alpha. By doing so, NOS can convert L-arginine into nitric oxide, thus releasing nitric oxide in the eyes.

To ensure the plasmid contains those two sequences, we conduct PCR with their primer separately. Fig. 1 shows that the plasmid contains both of the desired sequences.

In order to test the function of the T7 promoter, we transformed the plasmid into BL21(DE3). Next, to observe the effects of various IPTG concentrations on NOS expression, we performed SDS-PAGE with different IPTG concentrations. The bacteria is cultured for two hours and induced with IPTG for 12 hours. In Fig. 2 we can observe that the first to the third lane express a similar thin band. Meanwhile, we can see a thicker band in the fourth lane, which means it has a higher protein expression when the IPTG concentration is 1 mM.

Goal:

Control NOS production by IPTG inducible systems to produce more nitric oxide.

Achievements:

1. Measure the NOS activity in different IPTG concentration at different time

2. NOS kinetic test

As we decided to use an IPTG inducible system, we conducted an experiment to determine whether IPTG concentration and induction time can control the production of NO. Therefore, we test the IPTG system by using different concentrations and induced at different times. Here, we are using E. coli BL21(DE3) strain. As seen in Fig. 3, the nitric oxide production is both time dependent and IPTG dependent. Thereby, we can observe whether the IPTG inducible system can effectively produce nitric oxide in a certain induction time.

After confirming the production of NO can be induced by IPTG, we test the kinetic of NOS. With Fig.4, we cultured the bacteria for 12 hours and are induced with 0.1 mM IPTG for 2 hours. We then applied the homogenized bacteria and substrate to the cornea of the Porcine eye to observe whether the concentration of nitric oxide will increase in the eye or not. The substrate runs out within 20 min and produces approximately 1.6 nmol nitric oxide. Through the graph below, we can observe that when there is 1.6 nmol nitric oxide in the porcine eye, it will diffuse into the cornea and the concentration of nitric oxide will increase six times higher when it reaches aqueous humour.

We are taking advantage of the IPTG inducible system to control NOS expression, thus producing nitric oxide. From all the experimental results above, we have proved that our system is functioning well. Starting from NOS construction, confirm NOS expression, until NOS functional test, we have provided convincing results to support our ideas.

Goal:

Enhance biofilm production and improve the binding affinity of our engineered bacteria.

Achievements:

1. Construction of CsgD and CsgA

2. Run SDS-PAGE to confirm expression

3. Functional test to measure the amount of biofilm production and binding affinity

We are very grateful that one of our PIs, Professor Masayuki Hashimoto, from the Institute of Molecular Medicine, with generosity, was providing us a BW25113 wild type strain, BW25113 △CsgD, and PCA24N plasmid to supported our CsgD and CsgA experiment.

As a first step, we construct plasmid that only contains pLac-csgD on BW25113 and an improved version that contains NOS, csgD and csgA using gBlock provided by IDT. After that, we transform into E. coli BL21(DE3) strain. Then, we use PCR to confirm that our bacteria carried CsgD and CsgA. The expected results are shown in Fig.6.

Furthermore, we ran SDS-PAGE to check the protein expression of CsgD and CsgA. We culture the bacteria for 2 hours, then add IPTG to induce for 12 hours long, and adjust the OD600 to three. We expected to see CsgD protein size around 25 kDa and CsgA around 15 kDa. The results below have shown the expected size we want for CsgD and CsgA protein expression.

Next, for further confirmation, we use a congo red dye to see the curli expression. If there is a biofilm, the absorbance value at 500 nm will be higher. We use BW25113 as control, BW25113 with CsgD, and NOS-CsgA-CsgD on BL21(DE3). First, we culture for 2 hours and add IPTG to induce. After overnight culture, we add congo red to 1 ml of bacteria and leave it for 2 hours. Next, we centrifuge to separate the supernatant and pellet. By using a microplate reader, we can measure the absorbance value at 500 nm and normalized by 600 nm wavelength. Through the Fig.7 below, we can see that the amount of biofilm is increased after adding csgA.

After conducting several research, we found a common way for biofilm staining by using 96-well plate assay. We soon discovered that most of the experimental error came from the staining procedure and the steps of removing supernatant which decrease the accuracy. We try to remove the supernatant by hitting the plate to the table. As shown in Fig. 8, the data standard deviation is high.

However, the standard protocols to test biofilm formation cannot directly support our claims to increasing binding affinity. Therefore, we come up with a simple and quantifiable measurement protocol for evaluating biofilm binding ability. To observe the adherence of bacteria to the contact lens surface, we culture the bacteria for 2 hours then induced with IPTG. Then, we proceed into a 96-well plate and leave for 12 hours of incubation. Afterward, we used external forces to test the bacteria binding affinity by free falling an object - a book, from different heights and measuring the OD₆₀₀ value to determine its concentration. As the concentration increases, the bacteria will have a greater binding affinity.

The results show the bacteria overexpressing CsgA-CsgD can stand greater force. Thereby, we can prove that overexpressing CsgA-CsgD not only enhances biofilm production, but our bacteria can also bind properly to the contact lens.

Goal:

To prevent our engineered bacteria surviving outside the contact lens.

Achievements:

1. T7 polymerase

2. Successfully prove our bacteria will not survive without diaminopimelate (DAP)

We use the knockout strain WM3064 which deleted the gene dapA encoding for 4-hydroxy-tetrahydrodipicolinate synthase that is essential for cell wall synthesis^[1] and growth. For the growth, the strain WM3064 must rely on the supplement of diaminopimelate (DAP) in medium; in other words, the bacteria can’t survive without DAP. With this concept, we designed our kill switch for safety.

We transformed a plasmid carrying T7 RNA polymerase into WM3064 to control the expression of nos by the T7-lacI promoter. After that, we ran a SDS-PAGE to ensure the protein expression of nos under the T7-lacI promoter. The expected protein size of NOS is 40 kDa. As seen in Fig.14, our WM3064 with T7 RNA polymerase can successfully produce NOS.

In order to prove that E. coli WM3064 can’t survive without DAP , we spread our E. coli WM3064 with T7 RNA polymerase to different plates. As seen in this result (Fig.15), the bacteria can survive in the culture with exogenous DAP. When there is no more DAP supplied, the bacteria cannot survive.