Revision as of 13:42, 20 October 2019

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

Three strains expression on GFP and its control

Background

In Team NAWI_Graz 2019 characterization, we found that in normal growth/incubation condition (37 ^oC, LB agar) BBa_J364000-transformed Escherichia coli BL21 cells had the highest GFP expression in comparison to BL21 Star and Top 10 strains. In order to confirm that we have measured the GFP Flourescence of those three strains over 44 hours period.

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)
The broth became ligh green in color under natural light after around 14-18 hours of incubation time.

Figure 1: GFL Flourescence and OD600 Measurements with GFP containing E.coli strains and non GFP containing E.coli strains (control))

Figure 2: ~14 hours old GFP-transformed E. coli strains (Left: Top10, Middle: BL21, Right: BL21 Star)

Improvement

Group: iGEM Team UESTC-China 2019
Author: Qiuyun Huang, Zixin Wang

This year, we improved this reporter device (BBa_J364000) into a surface display system (BBa_K3034007) by fusing GFP with INPNC. 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.

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.

Quantitative detection of fluorescence

First, we cultured the bacteria overnight and adjusted them to the same OD600. We ultrasonic broken, centrifuged and respectively resuspend precipitation to measure the distribution of GFP in E.coli DH5α carrying BBa_J364000 and E.coli DH5α carrying BBa_K3034007.

Fig.1. 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. This was not in line 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. 2a). 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. 2b,2c). 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. 2c). The result is consistant with fact that we found in the literature[2] that the INPNC forms aggregates in the cell membrane.

Fig.2. The fluorescence microscopy of E.coli DH5α carrying BBa_J364000(a) and E.coli DH5α carrying BBa_K3034007 (b、c).

Conclusion

By fusing GFP with INPNC, we can implement the following improvements:

We upgraded this part into a surface display and report system, which can anchor GFP to the surface of E.coli and realize the function enhancement of the original part.
While GFP is used to report the expression of other enzymes, the system can also anchor other enzymes together with GFP to the bacterial surface to realize the surface display of certain enzymes and enhance the enzyme activity.

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.

@@ Line 78: / Line 78: @@
 ----
-We improved this reporter device into a surface presentation + reporting system (<html><a href='https://parts.igem.org/Part:BBa_K3034007'>BBa_K3034007</a></html>) by fusing GFP with INPNC so that the team could make reporter genes through GFP and anchor the target protein to the bacteria outer membrane for more applications.
+This year, we improved this reporter device (BBa_J364000) into a surface display system (<html><a href='https://parts.igem.org/Part:BBa_K3034007'>BBa_K3034007</a></html>) by fusing GFP with INPNC. 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.
-Ice nucleation protein (INP) is a secretory outer membrane protein from Pseudomomas syringae P. flurorescens and several other Gram—negative bacteria[1]. INP can anchor one or more "passenger proteins" to the outer membrane of ''E.coli'' DH5α. The fixation of exogenous proteins on the cell 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.
+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.
 ====Quantitative detection of fluorescence====
-We first cultured the bacteria overnight and made OD600 uniform. We ultrasonic broken, centrifuged and respectively resuspend precipitation to measure the distribution of GFP in ''E.coli'' DH5α carrying BBa_J364000 and ''E.coli'' DH5α carrying <html><a href='https://parts.igem.org/Part:BBa_K3034007'>BBa_K3034007</a></html> (Fig.1).
+First, we cultured the bacteria overnight and adjusted them to the same OD600. We ultrasonic broken, centrifuged and  respectively resuspend precipitation to measure the distribution of GFP in ''E.coli'' DH5α carrying BBa_J364000 and ''E.coli'' DH5α carrying <html><a href='https://parts.igem.org/Part:BBa_K3034007'>BBa_K3034007</a></html>.
 [[File:T_UESTC_China_Relative_FI.png|700px|thumb|center|'''Fig.1.''' The relative fluorescence intensity of ''E.coli'' DH5α carrying BBa_J364000 and ''E.coli'' DH5α carrying <html><a href='https://parts.igem.org/Part:BBa_K3034007'>BBa_K3034007</a></html>.
 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 after centrifugation. Moreover, the distribution of GFP in ''E.coli'' DH5α with <html><a href='https://parts.igem.org/Part:BBa_K3034007'>BBa_K3034007</a></html> was not significantly different from that in ''E.coli'' DH5α with BBa_J364000. But, the content of GFP in the broken ''E.coli'' DH5α with <html><a href='https://parts.igem.org/Part:BBa_K3034007'>BBa_K3034007</a></html> was higher than that in the ''E.coli'' DH5α with BBa_J364000.
+The results showed that both precipitation and supernatant contained relatively strong GFP. Moreover, the distribution of GFP in ''E.coli'' DH5α carrying <html><a href='https://parts.igem.org/Part:BBa_K3034007'>BBa_K3034007</a></html> was not significantly different from that in ''E.coli'' DH5α carrying BBa_J364000. This was not in line with our expectation, after analysis, it may be caused by incomplete ultrasonic broken of bacteria.
-Since the ''E.coli'' DH5α carrying <html><a href='https://parts.igem.org/Part:BBa_K3034007'>BBa_K3034007</a></html> expressed GFP, this indirectly indicated that INPNC was successfully expressed. However, the content of GFP in the ''E.coli'' DH5α precipitate (cell membrane) carrying <html><a href='https://parts.igem.org/Part:BBa_K3034007'>BBa_K3034007</a></html> was not significantly higher than that in the control group (with BBa_J364000). We hypothesized that INPNC was expressed but not highly active.
+Since the ''E.coli'' DH5α carrying <html><a href='https://parts.igem.org/Part:BBa_K3034007'>BBa_K3034007</a></html> expressed GFP, this indirectly indicated that INPNC was successfully expressed. However, the content of GFP in the ''E.coli'' DH5α precipitate (cell membrane) carrying <html><a href='https://parts.igem.org/Part:BBa_K3034007'>BBa_K3034007</a></html> 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, ''E.coli'' DH5α with BBa_J364000 (GFP) was observed to be rod-shaped and fluorescently filled with ''E.coli'' DH5α under a 40-fold microscope. The fluorescence of ''E.coli'' DH5α with <html><a href='https://parts.igem.org/Part:BBa_K3034007'>BBa_K3034007</a></html> (INPNC+GFP) was observed to be dotted and dispersed on the surface of ''E.coli'' DH5α. The results proved that INPNC was successfully expressed and functioned (Fig.2).
+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. 2a). The fluorescence of ''E.coli'' DH5α carrying <html><a href='https://parts.igem.org/Part:BBa_K3034007'>BBa_K3034007</a></html> (INPNC+GFP) was observed to be dotted and dispersed on the surface of ''E.coli'' (Fig. 2b,2c). 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 <html><a href='https://parts.igem.org/Part:BBa_K3034007'>BBa_K3034007</a></html> (INPNC+GFP) had fluorescence aggregation on one side of the ''E.coli'' surface (Fig. 2c). The result is consistant with fact that we found in the literature[2] that the INPNC forms aggregates in the cell membrane.
 [[File:T_UESTC_China_INPNC.png|700px|thumb|center|'''Fig.2.''' The fluorescence microscopy of ''E.coli'' DH5α carrying BBa_J364000(a) and ''E.coli'' DH5α carrying <html><a href='https://parts.igem.org/Part:BBa_K3034007'>BBa_K3034007</a></html> (b、c). ]]
-In addition, we also noticed that ''E.coli'' DH5α carrying <html><a href='https://parts.igem.org/Part:BBa_K3034007'>BBa_K3034007</a></html> (INPNC+GFP) had fluorescence aggregation on one side of the ''E.coli'' DH5α surface. The result is consistant with fact that we found in the literature[2] that the INPNC forms aggregates in the cell membrane.
 ====Conclusion====
-====Improvement and application====
 By fusing GFP with INPNC, we can implement the following improvements:
 # We upgraded this part into a surface display and report system, which can anchor GFP to the surface of ''E.coli'' and realize the function enhancement of the original part.
@@ Line 114: / Line 108: @@
 ====References====
-[1] Yang, X., Sun, S., Wang, H., & Hang, H. (2013). Comparison of autotransporter and ice nucleation protein as carrier proteins for antibody display on the cell surface of Escherichia coli. Prog Biochem Biophys, 40, 1209-19.
+[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] Lee, S. Y., Choi, J. H., & Xu, Z. (2003). Microbial cell-surface display. Trends in biotechnology, 21(1), 45-52.
+[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.