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.

Table 1...

Figure 4. Histogram of the fluorescence intensity of samples in Table 1...

According to the histogram above, we can see that Sample 1 (NO3-+ S4O62-) presents obvious higher fluorescence intensity than the rest three samples which means our design working. But we do realize that Sample 2 (S4O62-) also presents fluorescence than the blank control which confused us a lot. According to our design, if we only add S4O62- without NO3-, this AND gate wouldn’t allow to pass into the reporter (Part 3) as there is a closed door of the NO3- sensor (Part 1). Therefore, we thought there was something wrong in Part 1.

In order to figure out the reason, we did several literature research and find out that in Part 1 we didn’t build the NarX-NarL two-component regulator (Figure 5), which shall be the first one to sense nitrate and that is why Part 1 didn’t work fine as we expected.

Figure 5. Genetic construction design map...

Ref. Woo, Seung-Gyun, et al. "A designed whole-cell biosensor for live diagnosis of gut inflammation through nitrate sensing." Biosensors and Bioelectronics 168 (2020): 112523.

Attempt 2

Figure 6...

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 6), 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 7. Histograms of the fluorescence intensity of samples in Table 2...

Figure 8. Trend Contrast of the fluorescence intensity between that under OD600=0.8 and that under OD600=1...

According to the trend in Figure 8, 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 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]