Part:BBa_K3994010

PhrpL_amilGFP_IL10_PJ23104_ttrS

promoter

Usage and Biology

Background

nflammatory 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.

Construct design

This part is a coding sequence of amilGFP and IL10. amilGFP is a fluorescent protein. IL10 is an anti-inflammatory factor. These two factors were under PhrpL promoter. And this part is inserted into plasmid.

Figure 1. PhrpL_amilGFP_IL10_PJ23104_ttrS box...

Figure 2. The sequence map of PhrpL_amilGFP_IL10_PJ23104_ttrS...

Usage and Biology

It is the sequence of inducible promoter PhrpL. HrpR protein binds to HrpS protein forming a complex and then triggering the transcription of induced promoter PhrpL. It functions like a AND logic gate to open a gene circuit. The hrpL promoter (PhrpL) contains a functional UAS relative to the transcription start-site, to which purified HrpR, HrpS and HrpRS bind in electrophoretic mobility shift assays.

Usage and Biology

This is a coding sequence of IL10. This is a major immune regulatory cytokine that acts on many cells of the immune system where it has profound anti-inflammatory functions, limiting excessive tissue disruption caused by inflammation.

Proof of function

Attempt 1

Figure 3 Genetic Construction of IBD Distinguisher...

Sample 1: NO3- + S4O62-

Sample 2: S4O62-

Sample 3: NO3-

Sample 4: Blank Control

As shown above, there is obvious fluorescence in sample 1 and sample 2; for sample 3 and sample 4, there also a slight fluorescence produced in the tubes. In order to scientifically quantify the result, we used ELIASA to read the accurate fluorescence intensity, respectively.

Figure 3 Genetic Construction of IBD Distinguisher...

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 25
  Illegal XbaI site found at 175
  Illegal PstI site found at 13
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 2233
  Illegal NheI site found at 2256
  Illegal PstI site found at 13
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 31
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 25
  Illegal XbaI site found at 175
  Illegal PstI site found at 13
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 25
  Illegal XbaI site found at 175
  Illegal PstI site found at 13
  Illegal AgeI site found at 379
  
• 1000
  COMPATIBLE WITH RFC[1000]