Composite

Part:BBa_K1897028

Designed by: Keshiniy Madivannan   Group: iGEM16_NUS_Singapore   (2016-10-11)
Revision as of 17:26, 18 October 2016 by Phuongnguyen (Talk | contribs)


p70-33-sfGFP-Terminator

The construct contains one of the synthetic lldR promoters designed by team ETH-Zurich, IGEM 2015 (Part:BBa_K1847008) with a weaker RBS flanked by non-coding sequences (Part:BBa_K1897031), followed by a coding sequence of sfGFP (Part:BBa_K1897033) and a lambda t0 terminator (Part:BBa_K1897030).


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 78
    Illegal NheI site found at 101
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 589
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Usage and Biology

This is RIOT sensor 10, along with RIOT sensor 11 (Part:BBa_K1897029), which are designed to detect the increased level of L-lactate produced by tumor cells in the microenvironment. To demonstrate this functional proof of concept, we measured the level of sfGFP expression under the control of lactate sensors (Figure 3). Commercially available L-lactate solution was used to activate the sensors, hence induce the expression of sfGFP. Detailed working mechanism of RIOT sensors are shown in Figure 1.

Figure 1: Working mechanism of RIOT sensor.
(A) In the absence of lactate, lldR binds to two operators in the promoter region and inhibit the expression of a superfolded green fluorescent protein (sfGFP).
(B) In the presence of lactate, lactate binds lldR, preventing its binding to the operators. Consequently, sfGFP is expressed and the level of sfGFP can be quantified using fluorescence microscopy.
CAMr = Chloramphenicol resistant.

Construction of RIOT sensor 10: p70-33-sfGFP-Terminator

The design of RIOT sensors 10 and 11 is based on a shorter version of the promoter region for the wild-type lldPRD operon (Part:BBa_K1897037, derived from Part Part:BBa_K822000) and a modified version of this promoter, lldRO1-J23117-lldRO2 (Part:BBa_K1847008), which are hereafter referred to as “p62” and “p70”, respectively for convenient purpose. We received them from team ETH_Zurich 2015. We minimise the basal expression of our reporter by linking ribosomal binding site (RBS) of different strength with these promoters (Table 1). The efficiency of the medium RBS (Part:BBa_B0032) and the weak RBS (Part:BBa_B0033) are about 30% and 1% relative to the strong RBS (Part:BBa_B0034), respectively.

Table 1: Details of RIOT sensors containing a superfolded green fluorescent protein (sfGFP)

RIOT Sensor No. Construct Biobrick Details
10 p70-33-sfGFP-Terminator Part:BBa_K1897028 The construct contains one of the synthetic lldR promoters designed by team ETH-Zurich, IGEM 2015 (Part:BBa_K1847008) with a weak RBS (Part:BBa_B0033), followed by a CDS of sfGFP and a lambda t0 terminator.
11 p62-33-sfGFP-Terminator Part:BBa_K1897029 The construct contains a shorter version of the promoter region for the wild-type lldPRD operon (Part:BBa_K1897037) derived from Part Part:BBa_K822000, a weak RBS (Part:BBa_B0033), followed by a CDS of sfGFP and a lambda t0 terminator.
Figure 2: Construct 10: p70-33-sfGFP-Terminator was made using PCR overlap. The expected size, including Biobrick Prefix, Suffix and bases flanking restriction enzyme recognition sequences, is 1056 base pairs. Lane 1 marks the DNA ladder. Lane 4-7 are replicates of the same PCR reaction.

Characterization of RIOT sensor 10: p70-33-sfGFP-Terminator

The performance of RIOT sensor 10 and 11 was compared to other lactate sensors via the fold change in GFP expression when lactate concentration increased. The results showed that RIOT sensor 10, p70-33-sfGFP-Terminator (Part:BBa_K1897028) is more sensitive to small changes in lactate concentration (Figure 3). The p70-34-sfGFP-Terminator sensor containing lldRO1-J23117-lldRO2 promoter (Part:BBa_K1847008) with a strong RBS and the p62-34-sfGFP-Terminator sensor containing the natural promoter with a strong RBS, had relatively high basal expression, about 7 times higher than RIOT sensor 10. In addition, they did not show significant difference in the level GFP intensity when lactate concentration increased from 10-2 M to 10-3 M. Although RIOT sensor 11, p62-33-sfGFP-Terminator (Part:BBa_K1897029) had low basal expression, it did not respond well to the increase in lactate concentration.

Figure 3: Comparisons of sensitivity to changes in lactate concentration among RIOT sensors and other lactate sensors.
Overnight cultures of bacteria transformed with different sensors were diluted and incubated with various lactate concentration including 0 M, 10-3 M and 10-2 M. The best sensor is expected to have low basal expression to minimize false positive results while exhibiting sensitivity to small changes in lactate concentration.
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