Team:XMU-China/Project PSystem

A REASONABLE EXPLANATION OF MISFOLDING GFP UNDER QS OSCILLATION

In the project iGEM13_XMU-China, they couldn't get expected oscillation. However, this year iGEM14_XMU-China further investigated the reason of abnormal oscillation. We further reviewed SDS-PAGE analysis to confirm the circuit at protein level. The SDS-PAGE data is shown in Figure 1. Based on that, we made a reasonable assumption that the unexpected behavior of the LuxR promoter leads to the misfolding proteins Hence result in the abnormal oscillation result in the abnormal oscillation.

As the SDS-PAGE shows, a large amount of GFP-LVA and LuxI-LVA appear in pellet where misfolding proteins often exist. Both proteins directly affect the oscillation result. And it is critical to find out the reason for misfolding proteins. iGEM14_XMU-China made the following assumption:

The research^[1] reveals an unexpected behavior of Lux pR (BBa_R0062). In the absence of autoinducer 3OC6 (AHL), LuxR binds to plux (Lux pR) and activates backwards transcription (Figure 2).

The imperfect simplification of setting lux pL and Lux pR in the same direction: 

From the original design by Jeff Hasty, Lux pR and Lux pL were set in opposite directions (Figure 3A). In the absence of AHL, LuxR could activate backwards transcription of Lux pR leading to more expression of LuxR which was critical to meet the oscillation conditions. However, present literature did’t consider the backwards transcription which had effect on quorum sensing oscillation.

In the simplified design (Figure 3B), when LuxR activates the backwards transcription, RNA polymerase will be blocked by the terminators B0015. So this simplification will not perform as same as original design. Actually, the reverse terminated efficiency of B0015 is 0.295(CC)^[2] which may lead to leakage transcription. However, the correct sequence of GFP-LAA couldn’t be transcribed during the backwards transcription, even if the minus-strand of GFP-LAA could be transcribed, the sequence of the RNA is not in the right direction of GFP-LAA, hence incorrect amino acid sequences might be translated, resulting in misfolding GFP just as what the SDS-PAGE shows (Figure 1).

Because of the imperfect simplified design didn’t follow the original function completely, the abnormal oscillation was justifiable. Misfolding protein weren evidence to support our assumption.

iGEM14_XMU-China involved sequence comparison to investigate the difference between the original and the registry parts. We found that the original Lux pR had 20bp overlapping sequence with originalal Lux pR. There was a restriction enzyme cutting site (EcoR I) at the 56bp of original Lux pR (Figure 4).

Parts registry truncated the original Lux pR at 56bp to get the 55bp Lux pR (BBa_R0062). On the contrary, Lux pL (BBa_R0063) is longer than the original Lux pL, and at the end of BBa_R0063 is initial part of 41bp LuxR (BBa_C0062). Thus new problems arised—was the modification of original QS promoter reasonable? Did the modification result in the unexpected backwards transcription?

Quorum sensing system is so widely used in the synthetic biology, we thought it’s remarkable to make it clear. We highlighted the abnormal phenomenon of QS oscillation which might be caused by imperfect simplification for the very first time. We hope that more efforts could be made to figure out the interaction between QS oscillation parts.^[3]

References

1. Eckdahl T, Sawyer E M, Barta C, et al. Bacterial logic devices reveal unexpected behavior of frameshift suppressor tRNAs[J]. Interdisciplinary Bio Central, 2012, 4(1): 10.

2. http://parts.igem.org/Part:BBa_B0015

3. Danino T, Mondragón-Palomino O, Tsimring L, et al.A synchronized quorum of genetic clocks[J]. Nature, 2010, 463(7279): 326-330.