Measurement

Part:BBa_J364000

Designed by: Traci Haddock-Angelli   Group: Measurement_Lab   (2017-04-19)


Test Device 1 for the iGEM InterLab Study

This is a GFP expressing constitutive device for the 2017 iGEM InterLab study. It is called Test Device 1 for the study for easy reference.

This device is stored in pSB1C3 for the InterLab and is fully BioBrick compatible.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 705


Team NAWI_Graz 2019: GFP dynamics of transformed Escherichia coli strains in LB broth

Background

In Team NAWI_Graz 2019, we found that in normal growth/incubation condition (37oC, LB media + chloramphenicol) BBa_J364000-transformed Escherichia coli BL21 cells had the highest GFP expression in comparison to BL21 Star and Top 10 strains. Values were consistently higher for BL21 strain over the entire 44-hours measuring period.

Three strains expression on GFP and its control
Figure 1: GFL Flourescence Measurements with GFP containing E.coli strains and non GFP containing E.coli strains (control)). Remark: the green line represents all the GFP empty strains
Experimental Design

We used three different strains of transformed E. coli (Top10,BL21 and BL21 Star) for this study. Transformed strains were incubated in 15 mL LB broth 37 oC overnight. Next day they were used to inoculate a 50 mL LB to OD600=0.1. The cultures were then incubated at 37 oC, 140 rpm and samples were taken every 4 hours for 2 days to determine the green fluorescence protein (GFP) fluorescence intensity at 510 nm.

Result and Findings
  • There are significant differences of GFP expression in different strains of E. coli (Top10, BL21, BL21 Star)
  • Cultures turned light green under white light after around 14-18 hours of incubation time.


          Figure 2: GFL Flourescence and OD600 Measurements with GFP containing E.coli strains and non GFP containing E.coli strains (control))


Figure 3: at the 14h time point old GFP-transformed E. coli strains (Left: Top10, Middle: BL21, Right: BL21 Star)


Improvement


This year, we improved this reporter device (BBa_J364000) into a surface display system (BBa_K3034007) (Fig. 1) by fusing GFP with INPNC (BBa_K523008). The improved system can anchor the downstream protein of INPNC to the surface of bacteria, while the GFP is used as the reporter gene. So, other teams who have the need of surface display can insert their target gene into this system.

Fig.1. Schematic map of BBa_K3034007.

Ice nucleation protein (INP) is a secretory outer membrane protein from Pseudomomas syringae, P.flurorescens and several other Gram-negative bacteria. INP can anchor one or more "passenger proteins" to the outer membrane of bacteria. The fixation of exogenous proteins on the bacteria surface through INPNC can not only greatly improve the efficiency of enzymatic reaction, but also avoid the degradation of exogenous proteins by intracellular enzymes of host cells[1].

Besides, we added a segment of linker between INPNC and GFP to ensure that two adjacent domains do not sterically interfere with one another. In our experiments, without linker, GFP could not be properly expressed.

Quantitative detection of fluorescence

First, we cultured the bacteria overnight and adjusted them to the same OD600. We ultrasonic broke, centrifuged and respectively resuspend precipitation to measure the fluorescence intensity of GFP in E.coli DH5α carrying BBa_J364000 and E.coli DH5α carrying BBa_K3034007 (Fig. 2).

Fig.2. The relative fluorescence intensity of E.coli DH5α carrying BBa_J364000 and E.coli DH5α carrying BBa_K3034007. The relative fluorescence intensity= Fluorescence of precipitation/ (Fluorescence of supernatant+ Fluorescence of precipitation)×100%.

The results showed that both precipitation and supernatant contained relatively strong GFP. Moreover, the distribution of GFP in E.coli DH5α carrying BBa_K3034007 was not significantly different from that in E.coli DH5α carrying BBa_J364000. There are some differences with our expectation, after analysis, it may be caused by incomplete ultrasonic broken of bacteria.

Since the E.coli DH5α carrying BBa_K3034007 expressed GFP, this indirectly indicated that INPNC was successfully expressed. However, the content of GFP in the E.coli DH5α precipitate (cell membrane) carrying BBa_K3034007 was not significantly higher than the E.coli DH5α carrying BBa_J364000. We hypothesized that INPNC was expressed but the efficiency was not so high.

Microscopic observation

Next, we used fluorescence microscopy to see if the INPNC worked. E.coli DH5α carrying BBa_J364000 (GFP) was rod-shaped and the fluorescence was equably distributed in E.coli (Fig. 3a). The fluorescence of E.coli DH5α carrying BBa_K3034007 (INPNC+GFP) was observed to be dotted and dispersed on the surface of E.coli (Fig. 3b,3c). The results proved that GFP has apparently been anchored to the surface of the E.coli and INPNC was working.

In addition, we also noticed that E.coli DH5α carrying BBa_K3034007 (INPNC+GFP) had fluorescence aggregation on one side of the E.coli surface (Fig. 3c). The result is consistant with fact that we found in the literature[2] that the INPNC forms aggregates in the cell membrane.Thus, we are more clear that we have achieved our improvement.

Fig.3. The fluorescence microscopy of E.coli DH5α carrying BBa_J364000(a) and E.coli DH5α carrying BBa_K3034007 (b、c).


Conclusion

  1. We improved this part. GFP is an intracellular reporter gene, and we achieved the expression of GFP on the surface of E.coli.
  2. Based on the current results, there is space for further improvement in the efficiency of system expression, and the key point is that the activity of INPNC needs to be improved.
  3. Other teams who have the need of surface display can insert their target gene into this system. For example, the system can be applied to whole-cell biocatalysts, heavy metal contamination adsorption, antigen and antibody display, etc[1].

References

[1] Li mingya, & Lin chenshui. (2016). Ice crystal nuclear protein and its application in bacterial surface display technology. Amino acids and biological resources, 38(2), 7-11.

[2] Qiu, Y., Hudait, A., & Molinero, V. (2019). How Size and Aggregation of Ice-Binding Proteins Control Their Ice Nucleation Efficiency. Journal of the American Chemical Society, 141(18), 7439-7452.

[edit]
Categories
//cds/reporter/gfp
//classic/plasmid/measurement
Parameters
None