Difference between revisions of "Part:BBa K3994008"
 
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[[File:T--NOFLS YZ--BBa K3994001-Figure4.png|500px|thumb|center|Figure 6 The result of sequencing for plasmid pSU2718-part2...]]
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Sequencing feedback shows we have obtained the correct plasmids which is consistent with their DNA profiles.
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[[File:T--NOFLS YZ--BBa K3994001-Figure5.png|500px|thumb|center|Figure 7 The electrophoresis results of enzyme digestion (Middle) and PCR (Right)...]]
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Middle:
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Lane 1 and 2: Plasmid pSU2718-part2 digested by HindIII.
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Right:
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Lane 1: Part 1, PyeaR_HrpR, got by PCR method with size of 1293bp.
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This step is used to get the plasmids pSU2718-part2 digested by enzyme and gene PyeaR_HrpR by PCR method for later in the process. Therefore, channel 1 and 2 (Middle) were plasmids pSU2718--part2 digested by enzyme HindIII. And channel 1 (Right) was gene PyeaR_HrpR got by PCR. Clean-up the product to obtain pSU2718-part2 backbone and PyeaR_HrpR-fragment.
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T4 DNA ligase is used to connect pSU2718-part2 backbone and PyeaR_HrpR-fragment to get plasmid pSU2718-part2-part1.
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[[File:T--NOFLS YZ--BBa K3994001-Figure6.png|500px|thumb|center|Figure 8 E-coil having the desired pSU2718-part2-part1 (Left) and control (Right)...]]
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[[File:T--NOFLS YZ--BBa K3994001-Figure7.png|500px|thumb|center|Figure 9 The electrophoresis result of enzyme digestion...]]
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Lane 1: Recombinant plasmid pSU2718-part2-part1 without enzyme digestion.
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Lane 2: Recombinant plasmid pSU2718-part2-part1 digested by EcoR1.
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The result shows that we got the correct plasmid. And the plasmid was sent to sequence.
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[[File:T--NOFLS YZ--BBa K3994001-Figure8.png|500px|thumb|center|Figure 10 The result of sequencing for plasmid pSU2718-part2-part1...]]
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Sequencing feedback shows we have obtained the correct plasmids which is consistent with their DNA profiles.
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=== Proof of function ===
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==== Attempt 1 ====
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[[File:T--NOFLS YZ--BBa K3994001-Figure9.jpg|500px|thumb|center|Figure 11 Genetic Construction of IBD Distinguisher...]]
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Sample 1: NO3- + S4O62-
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Sample 2: S4O62-
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Sample 3: NO3-
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Sample 4: Blank Control
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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.
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[[File:T--NOFLS YZ--BBa K3994001-table1.png|500px|thumb|center|Table 1...]]
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[[File:T--NOFLS YZ--BBa K3994001-Figure10.png|500px|thumb|center|Figure 12. Histogram of the fluorescence intensity of samples in Table 1...]]
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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.
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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 13), which shall be the first one to sense nitrate and that is why Part 1 didn’t work fine as we expected.
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[[File:T--NOFLS YZ--BBa K3994001-Figure11.png|500px|thumb|center|Figure 13. Genetic construction design map...]]
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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.
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=== References ===
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==== 1 CDC -What is inflammatory bowel disease, available at: https://www.cdc.gov/ibd/what-is-ibd.htm ====
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==== 2炎症性肠病 百度百科available at: https://baike.baidu.com/item/%E7%82%8E%E6%80%A7%E8%82%A0%E7%97%85/5296499?fr=aladdin ====
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==== 3 Data and Statistics Inflammatory Bowel Disease (IBD) in the United StatesAvailable at: https://www.cdc.gov/ibd/data-statistics.htm ====
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==== 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. ====
  
  

Latest revision as of 09:30, 19 October 2021


PyeaR_HrpR


promoter

Profile

Name: PyeaR_HrpR

Base Pairs: 1223 bp

Origin: Synthetic

Properties: A coding sequence of HrpR protein.

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:

BBa_K3994001

Name: HrpR

Base Pairs: 948bp

Origin: Pseudomonas syringae, genome

Properties: A coding sequence of HrpR protein

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.

BBa_K3994000

Name: PyeaR

Base Pairs: 100bp

Origin: synthetic

Properties: A coding sequence of promoter ttB344.

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.



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.


Figure 7 The electrophoresis results of enzyme digestion (Middle) and PCR (Right)...


Middle:

Lane 1 and 2: Plasmid pSU2718-part2 digested by HindIII.

Right:

Lane 1: Part 1, PyeaR_HrpR, got by PCR method with size of 1293bp. This step is used to get the plasmids pSU2718-part2 digested by enzyme and gene PyeaR_HrpR by PCR method for later in the process. Therefore, channel 1 and 2 (Middle) were plasmids pSU2718--part2 digested by enzyme HindIII. And channel 1 (Right) was gene PyeaR_HrpR got by PCR. Clean-up the product to obtain pSU2718-part2 backbone and PyeaR_HrpR-fragment.

T4 DNA ligase is used to connect pSU2718-part2 backbone and PyeaR_HrpR-fragment to get plasmid pSU2718-part2-part1.


Figure 8 E-coil having the desired pSU2718-part2-part1 (Left) and control (Right)...


Figure 9 The electrophoresis result of enzyme digestion...


Lane 1: Recombinant plasmid pSU2718-part2-part1 without enzyme digestion.

Lane 2: Recombinant plasmid pSU2718-part2-part1 digested by EcoR1.

The result shows that we got the correct plasmid. And the plasmid was sent to sequence.

Figure 10 The result of sequencing for plasmid pSU2718-part2-part1...


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

Proof of function

Attempt 1

Figure 11 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 12. 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 13), which shall be the first one to sense nitrate and that is why Part 1 didn’t work fine as we expected.

Figure 13. 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.


References

1 CDC -What is inflammatory bowel disease, available at: https://www.cdc.gov/ibd/what-is-ibd.htm

2炎症性肠病 百度百科available at: https://baike.baidu.com/item/%E7%82%8E%E6%80%A7%E8%82%A0%E7%97%85/5296499?fr=aladdin

3 Data and Statistics Inflammatory Bowel Disease (IBD) in the United StatesAvailable at: https://www.cdc.gov/ibd/data-statistics.htm

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
    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]