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. 1. Confirmation of our construction by PCR. M: Marker; Lane 1: NOS (~2400 bp); Lane 2: CsgA-CsgD (~1500 bp). 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.

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 3923
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 1826
  Illegal SapI.rc site found at 2144