Difference between revisions of "Part:BBa K4115017"
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<b>Methods/Protocols</b><br> | <b>Methods/Protocols</b><br> | ||
− | 1. Before culturing, E.coli is stored at 4 degrees. Culture experiment group(pUC-SacC) and control group(pUC-Empty) in LB medium for 2.5h(OD600~0.6). Use these E.coli as the seeds for the following steps. | + | 1. Before culturing, E.coli is stored at 4 degrees. Culture experiment group(pUC-SacC) and control group(pUC-Empty) in LB medium for 2.5h(OD600~0.6). Use these E.coli as the seeds for the following steps. <br> |
− | 2. 1:100 add seeds to 100 ml M9 medium: 1.13g M9 salts, dissolved by 95ml ddH2O; 1M MgSO4 200ul; 1M CaCl2 10ul; 20%(m/v) sucrose solution 5ml (10 g/L as the final concentration). M9 salts solution, 1M MgSO4 and 1M CaCl2 are sterilized by autoclaving. 20% sucrose solution is sterilized by a filter. After autoclaving and cool at room temperature, add kanamycin. The final concentration of kanamycin is 30 mg/L. | + | 2. 1:100 add seeds to 100 ml M9 medium: 1.13g M9 salts, dissolved by 95ml ddH2O; 1M MgSO4 200ul; 1M CaCl2 10ul; 20%(m/v) sucrose solution 5ml (10 g/L as the final concentration). M9 salts solution, 1M MgSO4 and 1M CaCl2 are sterilized by autoclaving. 20% sucrose solution is sterilized by a filter. After autoclaving and cool at room temperature, add kanamycin. The final concentration of kanamycin is 30 mg/L.<br> |
3. Culture at 37 degrees, 220rpm. Use Nanodrop 100 to measure OD600 every 2h. If the OD600 is higher than 1.5, measure the OD600 after dilution. Every time take out 80ul liquids for tests. | 3. Culture at 37 degrees, 220rpm. Use Nanodrop 100 to measure OD600 every 2h. If the OD600 is higher than 1.5, measure the OD600 after dilution. Every time take out 80ul liquids for tests. | ||
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Revision as of 07:29, 3 October 2022
SacC (the extracellular sucrase gene)
The sequence of Zymomonas mobilis gene sacC that encodes the extracellular sucrase. Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Unknown
- 12INCOMPATIBLE WITH RFC[12]Unknown
- 21INCOMPATIBLE WITH RFC[21]Unknown
- 23INCOMPATIBLE WITH RFC[23]Unknown
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 437
Illegal NgoMIV site found at 696
Illegal AgeI site found at 418 - 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
The expression product is exhibited sucrase but not levansucrase activity,which means it behaves like a true sucrase. Heterologous expression of SacC aims to improve the utilization of sucrose in the medium by E. coli responsible for the synthetic production of biologics.
Cultivation, Experiment and Improvement Process
100 mL Fermentation E. coli DH5α with BBa_K4115017
![](/wiki/images/d/dc/SacCgrowthcurve1.png)
Methods/Protocols
1. Before culturing, E.coli is stored at 4 degrees. Culture experiment group(pUC-SacC) and control group(pUC-Empty) in LB medium for 2.5h(OD600~0.6). Use these E.coli as the seeds for the following steps.
2. 1:100 add seeds to 100 ml M9 medium: 1.13g M9 salts, dissolved by 95ml ddH2O; 1M MgSO4 200ul; 1M CaCl2 10ul; 20%(m/v) sucrose solution 5ml (10 g/L as the final concentration). M9 salts solution, 1M MgSO4 and 1M CaCl2 are sterilized by autoclaving. 20% sucrose solution is sterilized by a filter. After autoclaving and cool at room temperature, add kanamycin. The final concentration of kanamycin is 30 mg/L.
3. Culture at 37 degrees, 220rpm. Use Nanodrop 100 to measure OD600 every 2h. If the OD600 is higher than 1.5, measure the OD600 after dilution. Every time take out 80ul liquids for tests.