Part:BBa_K3598021

LacI_LacI promoter_T7 promoter_lac operator_RBS lib_sfGFP_T7 terminator

This part is an inducible expression system. The T7 promoter is regulated by lacI repressor through lacO operator to control the expression of sfGFP. When there is no inducer, sfGFP is not expressed and when the inducer IPTG is added, sfGFP is expressed and the fluorescence intensity increases. We construct a RBS library, this system is used for the construction and quantitative determination of our RBS libraty.

Experiments and Results

We have constructed a RBS library for the T7-LacI system, sfGFP was added for Quantitative identification. We began with randomly arranging six pairs in the system's RBS sequence TAAGTATAAGNNNNNNATAT, and verifying the resulting RBSs' strengths with RBS calculator. 15 RBSs were taken into our RBS library.

Figure 1. Overall design of BBa_K3598021

These RBSs are then integrated into the T7-LacI system through Goldengate, transformed into E. coli BL21 along with the original RBS as control group.

All the strains were cultivated in M9 medium with different concentration of IPTG, 25°C, for 20 hours. 10 μM culture were added to 190 μM PBS solution for fluorescence detection. From the result graph (Figure 2), we can see that the intensity of RBS varies from thousand to tens of thousand, covering a relatively wide range.

Figure 2. Quantitative identification of RBS library using sfGFP

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 2038
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1280
  Illegal BsaI.rc site found at 1270
  Illegal SapI.rc site found at 1303