Team:Heidelberg LSL/Notebook-15/03/2012

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iGEM-2012HS - LSL-Heidelberg iGEM-2012HS - LSL-Heidelberg



Notebook

Welcome to our notebook!

Here you will find the documentation of our laboratory work of the last few month in diary form. This notebook comprises the work in three phases:

            
Planning and
Development
            
            
Biosensor
Construction
              
            
Calibration and
Characterization
              

 

 

 

 

 

 

 

 

 

 

 

 

 

Biosensor Construction - 15/03/2012

  • The colony-PCR for the recA-GFP construct was repeated, now screening a number of 31 colonies. As control, again the amplicon of the original GFP biobrick was used.



Fig. 1 Repetition of colony-PCR for RecA-GFP construct. Clones #6, 18 and 26 were positive (shift in amplicon length of ~200 bp).

3 positive colonies were detected, as they show a shift in band size of ~ 200 bp as expected by the introduction of the recA promoter. Those were clones # 6, 18 and 26.

  • O/n LB-amp cultures were inoculated for clones #18 and #26.


  • Miniprep of the o/n cultures inoculated the day before for clones #2.3, 3.4 and 4.8 were done.
    • concentrations measured using Nanodrop are all in between 70 and 140 ng/┬Ál


  • Test digestions were performed in order to detect, whether the constructs would give the expected band patterns on the gel.
    • #2.3 with EcoRI/BglI; as control the original LacZ biobrick was digested EcoRI/BgII as well
    • #3.4 and #4.8 were digested NotI in order to see, whether an insert is present



Fig. 2 Test digestions for the constructs RecA-LacZ (#2.3), SulA-GFP (#3.4) and SulA-LacZ (#4.8). All constructs digested showed the right bands (left gel compared to virtual gel with expected bands on the right). Construct #2.3 gave the expected shift of the lowest band (arrow) compared to the LacZ-control construct (original biobrick BBa_K173004) due to the presence of the RecA promoter.