Team:XMU-China/Project PSystem

A reasonable explanation of misfolding GFP under QS oscillation

In the project of iGEM13 XMU-China, they can’t get expected oscillation. However, this year iGEM14 XMU-China further investigate the reason of abnormal oscillation. We further review SDS-PAGE analysis to confirm the circuit at protein level. The SDS-PAGE data is shown in Figure 1. Based on that, we make a reasonable assumption that the unexpected behavior of the LuxR Promoter leads to the misfolding proteins hence 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 make the following assumption:

The 2012 published paper^[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 are set in opposite directions (Figure 3A). In the absence of AHL, LuxR could activate backward transcription of Lux pR leading to more expression of LuxR which is critical to meet the oscillation conditions. However, present literature don’t consider the backwards transcription which have effect on quorum sensing oscillation.

In the simplified design (Figure 3B), when LuxR activates the backward transcription, RNA polymerase will be blocked by the terminators B0015. So that this simplification doesn’t 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 can’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 may be translated, resulting in misfolding GFP just as the SDS-PAGE shows (Figure 1).

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

iGEM14 XMU-China involved sequence comparison to investigate the difference between the original and the registry parts. We find that the original Lux pR has 20bp overlapping sequence with original Lux pR. There is a restriction enzyme cutting site (EcoR I) at the 56bp of original Lux pR (Figure 4).

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

Quorum sensing system is so widely used in the synthetic biology, we think it’s remarkable to make it clear. We highlight the abnormal phenomenon of QS oscillation which may 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.

http://www.ibc7.org/article/journal_v.php?sid=287

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.

http://www.nature.com/nature/journal/v463/n7279/full/nature08753.html