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).

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