Difference between revisions of "Part:BBa K1072000:Experience"

[http://2016.igem.org/Team:IISc_Bangalore]Team IISc Bangalore

Fluorescence/OD versus time curve for GFP expression under lux QS

While several quorum sensing parts exist in Parts Registry, relatively few examples exist of a full quorum-sensing sender and receiver in the same plasmid. BBa_K1072000 consists of both a Lux-based sender and a receiver module with GFPmut3 as the reporter. While the part has been shown to express GFPmut3 via microscopy, no growth curves of the same had been reported so far. Therefore, the IISc iGEM team 2016 decided to test the quorum sensing of this part in E coli BL21 (DE3).

Protocol

• Four 25 ml of LB media in conical flasks were inoculated from an overnight primary culture of E. coli BL21 (DE3) transformed with appropriate part (BBa_K1072000 for lux part and BBa_R0040 for negative control) in pSB1C3 (1 % inoculation, 37 degree celcius, 180 RPM).
• The cultures were grown in an orbit shaker incubator at 37 degree celcius, 180 RPM.
• 200 uL aliquots were taken from each conical flask every 2 hours and stored at 4 degree celcius.
• GFPmut3 fluorescence (Ex: 501 nm, Em: 511 nm) and OD600 of the samples were recorded on a plate reader (Tecan M2000 InfinitePro). (For detailed measurement conditions, please refer to the <raw data file>.)

Discussion

The primary culture had been grown for 12 hours before inoculation into the secondary and hence the cell densities had saturated. The AHL concentration in the medium per cell is expected to have been high; once the culture was diluted when inoculating into the secondary culture, the AHL concentration in the medium also reduced and hence, the rate of transcription from the luxI promoter and the rate of sfGFP production (it is under the luxI promoter) both decrease and are more than compensated for by cell division in the secondary culture, i.e., the number of copies of sfGFP per cell decreases because sfGFP synthesis is happening slower than cell division; this is reflected by the decrease in Flu/OD600. The Flu/OD600 values were initially decreasing up to around 6 hours; the culture's AHL concentration per cell started increasing after this point as the positive feedback in the AHL synthesis pathway has been initiated once the threshold AHL concentration is reached (happened at mid log in A. fischeri, the organism to which the pathway is native). As a result, the rate of production of sfGFP is higher than in the previous case (0-6 hours) and cell division also ceases shortly (saturation reached soon) and as a result, the sfGFP concentration per cell increases; this is indicated by the increase in Flu/OD600. The last data point alone showed a decrease, however, other than this point, the rest of the points shows an increasing trend, as expected.

A culture with just a pTet part (no quorum sensing) was intended to be used as a negative control, to see the change is Flu/OD600 due to the intrinsic fluorescence of the cells (due to NADPH, ATP, cytochromes etc, NOT due to sfGFP) and cell division reducing the concentration of these molecules per cell (as they are not being synthesised as fast as cells are dividing), reflected as Flu/OD600 decreasing. The data shows the expected decrease in Flu/OD600, however, the values are initially comparable to that of the cells expressing sfGFP, this indicates that the intrinsic fluorescence of the cells is changed very significantly by sfGFP expression in BBa_K1072000 transformants, indicating that the pTet transformed cells cannot be used as a negative control.

Results

Our experiment confirms the occurrence of a switch due to the QS system in BBa_1072000. This allows us to hypothesize practical applications of this system for cell density dependent processes, such as cell-density dependent protein over-expression for our project.