Part:BBa_I746902

pbad promoter driving 6-his tagged mut3 GFP

This part is used for purification of mut3 GFP via its 6-his tag. It is driven by the arabinose - sensitive pbad promoter. Induction should be with 1mM arabinose for overnight growth of a 50ml (or more) culture for purification the next day.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1205
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1880
  Illegal SapI site found at 961
  

User Experience by TecMonterrey_GDA

You can also see our process and results in the following pdg file

https://static.igem.org/mediawiki/2017/d/d3/CollabChihuahua.pdf

The synthesis of our construct took up all the IDT sponsorship, so we did some transformations from the kit of past parts, but none worked (nor the interlab parts). Since we as well as other teams had problems with the kit functioning properly, we asked Tec-Chihuahua Team help with some transformations with parts from previous years we wished to characterize, however we only focused on one:

BBa_I746902 pbad promoter driving 6-his tagged mut3 GFP

Excitation max: 501

Emission max: 511

To characterize and test it we made kinetics and compare agar plates of the transformed E. coli BL21 with chloramphenicol, two plates with the specified arabinose concentration and the sole plate without arabinose.

Photo of transformed cells E coli BL21 in agar with chloramphenicol and arabinose specified concentration 1mM in UV light. The fluorescence was bright green.

Photo of transformed cells E coli BL21 in agar only with chloramphenicol in UV light. The colonies appeared white, no green coloration observed.

To characterize it we made kinetics to measure time vs growth of transformed and not- transformed bacteria as well as measuring fluorescence of the GFPmut3 at different times and arabinose. The growth conditions were 37°C and 250 rpm.

The characterization took place with 4 kinetics. Each done with duplicate, and each time measured by triplicate, all kinetics had 20 ml of LB. The kinetics' characteristics are resumed in the following table:

**The previous growth of both cultures were 18 hours.

Results

Measurements were taken in a BIO-RAD SmartSpec Plus UV/Visible Spectrophotometer in 3 wavelengths, 600 nm for OD, 509 nm and 400 nm for GFP emission. Then comparisons and relationships between the results were established.

The “not-transformed” bacteria showed more growth on the fourth time Values differ mostly on the 4th hour, between the kinetics with L- arabinose and the ones without, possibly when GFPmut3 production may start.

All graphs and parameters:

Individual graphs:

For 1% v L-arabinose concentration:

For 0.5 % v of L-arabinose concentration:

For 0% v L-arabinose concentration:

For non-transformed bacteria, without arabinose and antibiotic: