Part:BBa_K3490001
IPTG inducible NOS, over-express csgD and csgA
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 each sequence’s primer separately. Fig.1 shows that the plasmid contains both of the desired sequences.
Fig. 2. SDS-PAGE of E.coli BL21(DE3) with different concentrations of IPTG. M: Marker; Lane 1: 0.1 mM IPTG; Lane 2: 0.05 mM IPTG; Lane 3: 0.025 mM IPTG; Lane 4: 1 mM IPTG. The arrow from top to bottom indicates NOS (~40kDa), CsgD (~24kDa), and CsgA (~17kDa).
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 nitric oxide. 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.
Fig. 3. NOS induced by IPTG with different concentrations at different induced times.
After confirming the production of nitric oxide 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.
Fig. 4. NOS activity at different working times to observe when the substrate will be depleted.
Fig. 5. The concentration of NO is six times higher after the bacteria and substrate is diffused into the porcin eye.
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 3923
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 1826
Illegal SapI.rc site found at 2144
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