Difference between revisions of "Part:BBa I15008"

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<partinfo>BBa_I15008 short</partinfo>
 
<partinfo>BBa_I15008 short</partinfo>
  
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One of two requisite genes required for the biosynthesis of phycocyanobilin from heme.
+
<span class='h3bb'>Sequence and Features</span>
 +
<partinfo>BBa_I15008 SequenceAndFeatures</partinfo>
  
 +
 +
<!-- Uncomment this to enable Functional Parameter display
 +
===Functional Parameters===
 +
<partinfo>BBa_I15008 parameters</partinfo>
 +
<!-- -->
 +
 +
===Description===
 +
One of two requisite genes required for the biosynthesis of phycocyanobilin from heme.
  
 
===Usage and Biology===
 
===Usage and Biology===
Line 11: Line 20:
 
==Additional Supplements==
 
==Additional Supplements==
 
The infomation below is updated by TJU_China of iGEM 2017.
 
The infomation below is updated by TJU_China of iGEM 2017.
 +
 +
===New infomation===
 +
We exchange the position of sequence window and the text.
 +
 +
The original part has a barcode after coding sequence. So we submit a new part [[Part:BBa_K2328062]] (without the barcode) for our composite parts. We will upload the data and infomation in all pages related to HO-1.
 +
 +
In addition, in order to make this part express better in hosts of our project (several intestinal bacteria), we optimize the codons of the part and submit another two new parts: [[Part:BBa_K2328003]] for E.coli, EHEC, Citrobacter rodentium, Lactococcus Iactis, Bacaeroides fragilis, Enterococcus faecalis and Clostridium difficile and [[Part:BBa_K2328004]] for Bifidobacterium longum. We do codon optimization for two obligate anaerobes just want to comfirm that this gene cannot work at all in obligate anarobes, which means HO-1 gene doesn't make any sense in these two bacteria, at least for now.
  
 
===New usage===
 
===New usage===
 
A novel far-red fluorescent protein evolved from APCα from Trichodesmium erythraeum, called smURFP, can covalently attaches a biliverdin (BV) chromophore without a lyase, unlike its precursor APC which should use an auxiliary protein known as a lyase
 
A novel far-red fluorescent protein evolved from APCα from Trichodesmium erythraeum, called smURFP, can covalently attaches a biliverdin (BV) chromophore without a lyase, unlike its precursor APC which should use an auxiliary protein known as a lyase
−
to incorporate phycocyanobilin. In addition, phycocyanobilin (PCB) is
+
to incorporate phycocyanobilin. In addition, phycocyanobilin (PCB) is synthesized from BV, and PCB do not exists in mammals but BV does. So BV is better as a chromophore in some way, along with smURFP.
  
−
===New infomation===
+
In our projext, we use HO-1 gene for two task: one is to produce BV in E.coli BL21, the other is to be a element in co-expression system (with fluorescent protein smURFP).
−
The original part has a barcode after coding sequence. So we submit a new part [[Part:BBa_K2328062]] (without the barcode) for our composite parts. But we will upload all the data and infomation in both pages.  
+
  
−
In addition, in order to make this part express better in hosts of our project (several intestinal bacteria), we optimize the codons of the part and submit another two new parts: [[Part:BBa_K2328003]] for E.coli, EHEC, Citrobacter rodentium, Lactococcus Iactis, Bacaeroides fragilis, Enterococcus faecalis and Clostridium difficile and [[Part:BBa_K2328004]] for Bifidobacterium longum. We do codon optimization for two obligate anaerobes just want to comfirm that this gene cannot work at all in obligate anarobes, which means HO-1 gene doesn't make any sense in these two bacteria, at least for now.
+
The sequence was taken by the St Andrews iGEM 2020 team and subject to the IDT codon optimisation tool. As a result of optimisation, two illegal restriction sites were introduced to the part and then removed to allow the part to be used at RFC[10] and RFC[1000] standard with codon optimisation (<partinfo>BBa_K3634007</partinfo>)
  
 
===Results===
 
===Results===
−
We use this part to prduce Biliverdin (BV) in E.coli BL21. For the subsequent experiments, we use our codon-optimized gene to express HO-1 in facultative anarobes.
+
:'''I. BV production'''
 +
 
 +
We use this part to prduce Biliverdin (BV) in E.coli BL-21. As shown in the picture, we can see the obvious change if medium in color.
 +
<p style="text-align: center;">
 +
    https://static.igem.org/mediawiki/parts/3/36/BV_production.png<br>
 +
'''Figure 1.'''  The result after induction, the upper one is the control group, and the inferior one is the experimental group.<br>
 +
</p>
 +
 
 +
----
 +
 
 +
:'''II. Co-expression with smURFP'''
 +
 
 +
Plasmid pET28b with smURFP and HO-1 gene were transformed into E. coli BL-21. We used this induced bacteria to confirm the fluorescence and data showed a relatively high value, as shown in table 1.
 +
<p style="text-align: center;">
 +
    https://static.igem.org/mediawiki/parts/e/e5/Microplate_Reader.png<br>
 +
'''Table 1.'''  Result of Microplate Reader in the black 96-well plate. Tube 1 and 2 are experimental group, and tube 3 is the control group.<br>
 +
</p>
 +
 
 +
 
 +
Then laser confocal microscopy was use to observe these bacteria, activate light of 640nm was used, as shown in Figure 2.
 +
 
 +
<p style="text-align: center;">
 +
    https://static.igem.org/mediawiki/parts/f/f1/Confocal.jpg<br>
 +
'''Figure 2.'''  The result after induction, the upper one is the control group, and the inferior one is the experimental group.<br>
 +
</p>
 +
 
 +
 
 +
After tests <i>in vitro</i>, we used this engineered bacteria for experiments <i>in vivo</i>. Utilizing Animal imaging system, we consistently observed the fluorescence emitted from the bacteria in mices' gut. The result successfully showed that our system was executable and excellent. And smURFP has very competible persistence and penetrability.
 +
 
 +
<p style="text-align: center;">
 +
    https://static.igem.org/mediawiki/2017/8/89/INVIVO.png<br>
 +
'''Figure 8.'''  The fluorescent inensity after doing intragastric administration for 5.5h.The left is the control one, the right is the experimental one.<br>
 +
</p>
 +
 
 +
For other experiments for some intestinal bacteria, we use our codon-optimized gene to express HO-1 in facultative anarobes.
 +
 
 +
----
 +
 
 +
 
 +
----
  
 
===Reference===
 
===Reference===
−
[1]  
+
[1] Rodriguez EA, Tran GN, Gross LA, Crisp JL, Shu X, Lin JY, Tsien RY. A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein. Nat Methods. 2016 Sep;13(9):763-9.
  
−
<span class='h3bb'>Sequence and Features</span>
+
[2] HO-1 I (without a barcode) [[Part:BBa_K2328062]]
−
<partinfo>BBa_I15008 SequenceAndFeatures</partinfo>
+
  
 +
[3] HO-1 II codon-optimized for intestinal bacteria [[Part:BBa_K2328003]]
  
−
<!-- Uncomment this to enable Functional Parameter display
+
[4] HO-1 III codon-optimized for Bifidobacterium longum [[Part:BBa_K2328004]]
−
===Functional Parameters===
+
−
<partinfo>BBa_I15008 parameters</partinfo>
+
−
<!-- -->
+

Latest revision as of 14:54, 9 August 2020

heme oxygenase (ho1) from Synechocystis

Sequence and Features


Barcodes are discontinued, but one was appended to the sequence of this part. Composite parts using this part will include the barcode. More ...

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Description

One of two requisite genes required for the biosynthesis of phycocyanobilin from heme.

Usage and Biology

ho1 oxidizes the heme group using a ferredoxin cofactor, generating biliverdin IXalpha and representing the first of two steps in phycocyanobilin (PCB) biosynthesis. PCB associates with Cph8, creating a light responsive protein complex. Functions in tandem with BBa_I15009 for PCB biosynthesis. PCB then associates with Part:BBa_I15010, a light responsive Cph8/EnvZ fusion protein.

Additional Supplements

The infomation below is updated by TJU_China of iGEM 2017.

New infomation

We exchange the position of sequence window and the text.

The original part has a barcode after coding sequence. So we submit a new part Part:BBa_K2328062 (without the barcode) for our composite parts. We will upload the data and infomation in all pages related to HO-1.

In addition, in order to make this part express better in hosts of our project (several intestinal bacteria), we optimize the codons of the part and submit another two new parts: Part:BBa_K2328003 for E.coli, EHEC, Citrobacter rodentium, Lactococcus Iactis, Bacaeroides fragilis, Enterococcus faecalis and Clostridium difficile and Part:BBa_K2328004 for Bifidobacterium longum. We do codon optimization for two obligate anaerobes just want to comfirm that this gene cannot work at all in obligate anarobes, which means HO-1 gene doesn't make any sense in these two bacteria, at least for now.

New usage

A novel far-red fluorescent protein evolved from APCα from Trichodesmium erythraeum, called smURFP, can covalently attaches a biliverdin (BV) chromophore without a lyase, unlike its precursor APC which should use an auxiliary protein known as a lyase to incorporate phycocyanobilin. In addition, phycocyanobilin (PCB) is synthesized from BV, and PCB do not exists in mammals but BV does. So BV is better as a chromophore in some way, along with smURFP.

In our projext, we use HO-1 gene for two task: one is to produce BV in E.coli BL21, the other is to be a element in co-expression system (with fluorescent protein smURFP).

The sequence was taken by the St Andrews iGEM 2020 team and subject to the IDT codon optimisation tool. As a result of optimisation, two illegal restriction sites were introduced to the part and then removed to allow the part to be used at RFC[10] and RFC[1000] standard with codon optimisation (BBa_K3634007)

Results

I. BV production

We use this part to prduce Biliverdin (BV) in E.coli BL-21. As shown in the picture, we can see the obvious change if medium in color.

BV_production.png
Figure 1. The result after induction, the upper one is the control group, and the inferior one is the experimental group.

II. Co-expression with smURFP

Plasmid pET28b with smURFP and HO-1 gene were transformed into E. coli BL-21. We used this induced bacteria to confirm the fluorescence and data showed a relatively high value, as shown in table 1.

Microplate_Reader.png
Table 1. Result of Microplate Reader in the black 96-well plate. Tube 1 and 2 are experimental group, and tube 3 is the control group.


Then laser confocal microscopy was use to observe these bacteria, activate light of 640nm was used, as shown in Figure 2.

Confocal.jpg
Figure 2. The result after induction, the upper one is the control group, and the inferior one is the experimental group.


After tests in vitro, we used this engineered bacteria for experiments in vivo. Utilizing Animal imaging system, we consistently observed the fluorescence emitted from the bacteria in mices' gut. The result successfully showed that our system was executable and excellent. And smURFP has very competible persistence and penetrability.

INVIVO.png
Figure 8. The fluorescent inensity after doing intragastric administration for 5.5h.The left is the control one, the right is the experimental one.

For other experiments for some intestinal bacteria, we use our codon-optimized gene to express HO-1 in facultative anarobes.


Reference

[1] Rodriguez EA, Tran GN, Gross LA, Crisp JL, Shu X, Lin JY, Tsien RY. A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein. Nat Methods. 2016 Sep;13(9):763-9.

[2] HO-1 I (without a barcode) Part:BBa_K2328062

[3] HO-1 II codon-optimized for intestinal bacteria Part:BBa_K2328003

[4] HO-1 III codon-optimized for Bifidobacterium longum Part:BBa_K2328004