Difference between revisions of "Part:BBa K1124011"

 
 
(3 intermediate revisions by one other user not shown)
Line 1: Line 1:
−
 
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K1124011 short</partinfo>
 
<partinfo>BBa_K1124011 short</partinfo>
  
−
hpaBC operon (+RBS(natural))
+
===Usage and Biology===
  
 +
This part consists of ''hpaB'' and ''hpaC'' genes, and is called ''hpaBC''
 +
The products of these genes, HpaB and HpaC, together form an enzyme 4-hydroxyphenylacetate 3-hydroxylase. This enzyme hydroxylates L-tyrosine to produce L-DOPA ''in vivo''.
  
−
<!-- Add more about the biology of this part here
+
It is known that hpaBC overexpression leads to increase of L-DOPA production in ''E. coli''.
−
===Usage and Biology===
+
We designed L-DOPA synthesis device([[Part:BBa_K1124107]]) by combining this part and tyrosine synthesis device([[Part:BBa_K1124106]]).
  
−
<!-- -->
 
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K1124011 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1124011 SequenceAndFeatures</partinfo>
 +
 +
=SMS_Shenzhen test this part in 2020 =
 +
To examine the efficiency of HpaBC-WT, we transform pHpaBC-WT  into a DH10b strain which is integrated with T7RNAP constitutive expression unit，which  confirm the practicability of our fermentation system.
 +
==Deteals==
 +
===Experimental setup===
 +
-  we first prepare DH10b[1], a strain that is integrated with T7RNAP constitutive expression unit, as component cell. <br>
 +
- Then we transform HpaBC-WT and HpaBC-SMS to component cells, respectively.<br>
 +
- The monoclonals are later overnight cultivated, and is then inoculated into fresh LB medium with a proportion of 1:200.<br>
 +
-  The samples are then cultivated for 48h, in 37℃, 220rpm. Samples are taken out, after the 48-hours-long cultivation, to measure for its L-dopa concentration.<br>
 +
-  The detailed description of measurement is illustrated below<br>
 +
===Results===
 +
We culture our strains overnight and find medium turning dark.<br>
 +
[[File:T--SMS_Shenzhen--1.png|600px|thumb|center|overnight cultivated bacteria culture]]<br>
 +
To explore the best substrate concentration for L-Dopa production, In this test, leveled concentrations of the substrate, tyrosine, (0mM, 0.03mM, 0.3mM, 1.5mM, 3mM, 4.5mM, 6mM, 9mM) are prepared. We then inoculate Bacteria in 7 mL LB medium and take for 500μL each time. They are taken at specific points: 30h, 39h, 42h, 50h. We use microplate reader to measure its absorbance under OD 400, and we determined the production of L-Dopa with standard curve of levodopa.<br>
 +
[[File:T--SMS_Shenzhen--2.png|600px|thumb|center|Exploration on the best fit for substrate concentration--the process of L-dopa measurement]]<br>
 +
The results are listed below.<br>
 +
[[File:T--SMS_Shenzhen--3.png|600px|thumb|center|Modeling on the HpaBC enzymatic reaction]]<br>
 +
The following column is about levodopa concentration in 3mM samples cultured for 42h.<br>
 +
[[File:T--SMS_Shenzhen--4.png|600px|thumb|center|Changes before and after the codon optimization of HpaBC]]<br>
 +
We can observe from the graph that, after codon optimization, HpaBC has presented higher biological activity. In the cultivation where the concentration of tyrosine is 3mM, after 42 hours of cultivation, the production of levodopa in HpaBC-WT is 0.88 mol/L; in the cultivation of HpaBC-SMS, the production is 1.12 mol/L. We thus chose HpaBC-SMS for the following experiments.<br>
 +
 +
  
  

Latest revision as of 23:08, 27 October 2020

hpaBC (+RBS(natural))

Usage and Biology

This part consists of hpaB and hpaC genes, and is called hpaBC The products of these genes, HpaB and HpaC, together form an enzyme 4-hydroxyphenylacetate 3-hydroxylase. This enzyme hydroxylates L-tyrosine to produce L-DOPA in vivo.

It is known that hpaBC overexpression leads to increase of L-DOPA production in E. coli. We designed L-DOPA synthesis device(Part:BBa_K1124107) by combining this part and tyrosine synthesis device(Part:BBa_K1124106).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 97
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1877

SMS_Shenzhen test this part in 2020

To examine the efficiency of HpaBC-WT, we transform pHpaBC-WT into a DH10b strain which is integrated with T7RNAP constitutive expression unit，which confirm the practicability of our fermentation system.

Deteals

Experimental setup

- we first prepare DH10b[1], a strain that is integrated with T7RNAP constitutive expression unit, as component cell.
- Then we transform HpaBC-WT and HpaBC-SMS to component cells, respectively.
- The monoclonals are later overnight cultivated, and is then inoculated into fresh LB medium with a proportion of 1:200.
- The samples are then cultivated for 48h, in 37℃, 220rpm. Samples are taken out, after the 48-hours-long cultivation, to measure for its L-dopa concentration.
- The detailed description of measurement is illustrated below

Results

We culture our strains overnight and find medium turning dark.

overnight cultivated bacteria culture

To explore the best substrate concentration for L-Dopa production, In this test, leveled concentrations of the substrate, tyrosine, (0mM, 0.03mM, 0.3mM, 1.5mM, 3mM, 4.5mM, 6mM, 9mM) are prepared. We then inoculate Bacteria in 7 mL LB medium and take for 500μL each time. They are taken at specific points: 30h, 39h, 42h, 50h. We use microplate reader to measure its absorbance under OD 400, and we determined the production of L-Dopa with standard curve of levodopa.

Exploration on the best fit for substrate concentration--the process of L-dopa measurement

The results are listed below.

Modeling on the HpaBC enzymatic reaction

The following column is about levodopa concentration in 3mM samples cultured for 42h.

Changes before and after the codon optimization of HpaBC

We can observe from the graph that, after codon optimization, HpaBC has presented higher biological activity. In the cultivation where the concentration of tyrosine is 3mM, after 42 hours of cultivation, the production of levodopa in HpaBC-WT is 0.88 mol/L; in the cultivation of HpaBC-SMS, the production is 1.12 mol/L. We thus chose HpaBC-SMS for the following experiments.