Part:BBa_K3994009

PttB344_HrpS_PJ23105_ttrR

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. It can sense S4O62- to release the substance S

Construct design

HrpS is a key functional factor under PttB344 promoter (Figure 1). And this sequence is inserted into plasmid. Its sequence is shown in Figure 2.

Figure 1. Boolean gate logic circuit/AND circuit construction...

Figure 2. PttB344_HrpS_PJ23105_ttrR, sensing S4O62- to release the substance S...

The profiles of every basic part are as follows:

Usage and Biology

This codes for HrpS 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 PttB344. PttrB185-269 is a minimal TtrR activated promoter when TtrR is phosphorylated by TtrS after TtrS sensing tetrathionate.

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.

Figure 6 The result of sequencing for plasmid pSU2718-part2...

Sequencing feedback shows we have obtained the correct plasmids which is consistent with their DNA profiles.

Proof of function

Attempt 1

Figure 7. Genetic Construction of IBD Distinguisher...

Sample 1: Negative Control (E. coli)

Sample 2: Positive Control (E. coli/amilGFP)

Sample 3: E. coli/pUC-57_Part 3

Sample 4: E.coli/pSU2718-Part 1_Part 2+pUC-57_Part 3

In order to further analyze the performance of Part 2, we also designed the control groups as showing above where Sample 1 and Sample 2 were presented as the comparison of the fluorescence phenomenon. We can see that there was no fluorescence in Sample 3 (the report part, Part 3). According to the result of Sample 3 and Sample 4 (Figure 7), we could infer that the AND gate design works (HrpR_HrpS, PhrpL) as the Part 3 didn’t respond when there is no Part 1 and Part 2. Of course, we also repeated the experiments and measured the fluorescence intensity by ELIASA. The data is given below.

Table 2...

Figure 8. Histograms of the fluorescence intensity of samples in Table 2...

Figure 9. Trend Contrast of the fluorescence intensity between that under OD600=0.8 and that under OD600=1...

According to the trend in Figure 9, the fluorescence intensity of pUC57-Part 3 presents the similar level as that of blank control and group NO3-, which means the “gate keeper” promoter, PhrpL works well and this conclusion also back up the conclusion that the AND gate design works well (HrpR_HrpS, PhrpL).

As the group S4O62 did present higher fluorescence intensity than that of group NO3- whenever OD600 equals to 0.8 or 1, it indicates that Part 2 and Part 3 both works well as Part 2 did show “green light” to the thiosulfate to finally “light” the Part 3.

References

4. Wang, Baojun, et al. "Engineering modular and orthogonal genetic logic gates for robust digital-like synthetic biology." Nature communications 2.1 (2011): 1-9.

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 839
  Illegal NheI site found at 862
  Illegal PstI site found at 13
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1893
  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 553
  Illegal AgeI site found at 1106
  Illegal AgeI site found at 1112
  Illegal AgeI site found at 1247
  
• 1000
  COMPATIBLE WITH RFC[1000]