Part:BBa_K3994008

PyeaR_HrpR

promoter

Usage and Biology

Background

Inflammatory bowel disease (IBD) is a chronic intestinal inflammatory disease of unknown etiology, including ulcerative colitis (CD) and Crohn’s disease (CD). This chronic disease, which is prone to repeated deterioration, currently lacks unified diagnostic and treatment standards, and is posing a great threat to public health. Drug therapy (anti-inflammatory drugs) is the preferred treatment for IBD. However, studies in the past 10 years have found that 30-50% of IBD patients do not respond to anti-TNF treatment. In addition, after long-term use of anti-inflammatory drugs, the patient's intestinal microbial status changes over time, and the effect may be lost due to drug resistance. Therefore, we need to seek help from other treatments for IBD. HrpR protein binds to HrpS protein forming a complex and then triggering the transcription of promoter hrpL. This part can sense NO3- to release the substance R.

Construct design

HrpR is a key functional factor under PyeaR promoter (Figure 1). And this part is inserted into plasmid. Its sequence is shown in Figure 2

Figure1 PyeaR_HrpR box...

Figure 2. The sequence map of PyeaR_HrpR, sensing NO3- to release the substance R...

The profiles of every basic part are as follows:

Usage and Biology

This codes for HrpR protein. HrpR protein binds to HrpS protein forming a complex and then triggering the transcription of promoter hrpL. This functions like a AND logic gate.

Usage and Biology

This is a sequence of PyeaR. It is a promoter. In our group, HrpR is under this promoter.

Experimental approach

Recombinant Plasmid Construction

Figure 3. The electrophoresis results of enzyme digestion and PCR...

Lane 1 and 2: Plasmid pSU2718-p15A digested by Xba1 and BamH1.

Lane 3: Part 2, PttB344_HrpS_PJ23105_ttrR, got by PCR method with size of 2060bp.

This step is used to get the plasmids pSU2718-p15A digested by enzyme Xba1 and BamH1and gene PttB344_HrpS_PJ23105_ttrR by PCR method for later in the process. Therefore, channel 1 and 2 were plasmids pSU2718-p15A digested by enzyme Xba1 and BamH1. And channel 3 were gene PttB344_HrpS_PJ23105_ttrR got by PCR. Clean-up the product to obtain pSU2718-p15A backbone and PttB344_HrpS_PJ23105_ttrR-fragment. T4 DNA ligase is used to connect pSU2718-p15A backbone and PttB344_HrpS_PJ23105_ttrR-fragment to get plasmid pSU2718-part2.

Figure 4 E-coil having the desired pSU2718-part2 (Left) and control (Right)...

Figure 5. The electrophoresis result of enzyme digestion identification...

Figure 5. The electrophoresis result of enzyme digestion identification...

Lane 1: Plasmid pSU2718-p15A digested by EcoR1.

Lane 2 to 5: Recombinant plasmid pSU2718-part2 digested by EcoRI. We got two bands with size of 2803bp and 1544bp. The results show that we got the correct plasmid. And the plasmid was sent to sequence.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 669
  Illegal AgeI site found at 142
  Illegal AgeI site found at 256
  Illegal AgeI site found at 262
  Illegal AgeI site found at 901
  
• 1000
  COMPATIBLE WITH RFC[1000]