Difference between revisions of "Part:BBa K3628025"
Bryan Zeng (Talk | contribs) |
|||
| Line 3: | Line 3: | ||
<partinfo>BBa_K3628025 short</partinfo> | <partinfo>BBa_K3628025 short</partinfo> | ||
| − | + | =Description= | |
| − | + | This part codes HpaBC-wild type, contains [[Part:BBa_K3628009|HpaB]], [[Part:BBa_K3628010|HpaC]]and [[Part:BBa_K3628013|RBS1 between HpaB and HpaC]] | |
| − | + | =Usage and Biology= | |
| − | + | ===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 02:25, 28 October 2020
HpaBC
Description
This part codes HpaBC-wild type, contains HpaB, HpaCand RBS1 between HpaB and HpaC
Usage and Biology
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.
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.
The results are listed below.
The following column is about levodopa concentration in 3mM samples cultured for 42h.
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.