Team:Aalto-Helsinki/Practice project

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Practice project: Testing RBSs

Introduction

In synthetic biology it is important to produce foreign compounds in a host. One aspect of this is constructing a synthetic genome and choosing the best compatible parts to have enough yield of a desired product.

A ribosomal binding site (RBS) is a location in a mRNA which a ribosome recognizes and binds to, thus initiating translation. The RBSs are defined by the efficiency with which they bind to ribosomes. Thus the strong RBSs bind more efficiently than the medium or the weak RBSs. The most product is formed when host cells are big and physically stable. According to Ceroni et al. [1], strong RBSs affect cell growth and eventually lower the yield of a wanted protein compared to weaker RBSs. The reason behind this is that the strong RBSs reduce significantly the translation of endogenous mRNAs which are needed for cell growth.

Before we started our actual project of producing propane in the lab, we thought that it would be interesting to test if weaker RBSs actually improve protein yield. By doing this, we could also get some practice of the common lab procedures.

Our goal was to create a construct using BioBricks to test RBSs in the following order: a T7 promoter, a RBS (varies; strong, medium or weak), a blue chromoprotein and a terminator. Table 1 shows more information about our construct parts.

Construct	Definition	Link (if BioBrick)	Size
AH001	Blue Chromoprotein	BBa_K592009	669
AH002	T7 promoter	BBa_I716106	29
AH003	Strong RBS	BBa_B0030	29
AH004	Medium RBS	BBa_B0032	13
AH005	Weak RBS	BBa_B0031	14
AH006	Terminator	BBa_B0015	129
AH007	AMP backbone	pSB1A3	2155
AH008	KAN backbone	pSB1K3	2204
AH009	CAM backbone	pSB1C3	2070
AH011	Blue chromoprotein + Ter in AH008		3002
AH013	T7 + Strong RBS in AH008		58
AH014	T7 + Medium RBS in AH008		42
AH015	T7 + Weak RBS in AH008		43
AH016	T7 + Strong RBS + Blue + Ter in AH009		2926
AH017	T7 + Medium RBS + Blue + Ter in AH009		2910
AH018	T7 + Weak RBS + Blue + Ter in AH009		2911

Table 1: Our construct parts to test RBSs.

Methods

Our idea was to create constructs with the three-antibiotic (3A) assembly method. After the 3A assembling we checked if our plasmids had the correct insert by restricting them (to avoid super-coil plasmids) and running them in an agarose gel, usually dyed with Ethidium Bromide.

The constructs had a T7 promoter, so we had to use a BL21 (DE3) strain to induce transcription with IPTG. Then we measured the absorption of the blue chromoprotein via spectrophotometer with 588 nm. The results of the measurement would have specified what kind of RBS is the best one as the spectrophotometer would tell us which RBS resulted in the best production yield of the blue chromoprotein.

The detailed descriptions of our procedures and methods can be found on the Protocols page.

Results

Our spectrophotometer results indicated that blue chromoprotein was not translated at all. After a while, we inspected our protocols and procedures thoroughly and finally had an answer to why it did not work: our 3A assembling was flawed, as shown in Figure 1. The main problem is that in the assembled plasmids possible the blue chromoprotein gene is before the T7 promoter and RBS so the blue chromoprotein gene wont be transcripted.

Figure 1: BioBrick 3A Assembly. The left side shows how we could have got working plasmids following correctly the method. The right side shows the best case of a scenario of our attempt of the 3A assembly. For example, it is more propable that only terminator ligate with the KAN backbone without blue chromoprotein inserts.

What we have learned

In the end, we did not create constructs that we were expecting, but we have learned so much about lab procedures, for example different ligation methods, and the importance of thorough planning. If we had checked carefully our restriction plans with instructors, we believe that we would have got different results and also saved time. At least, we understand now what went wrong. To make sure that the same thing does not happen with the propane production and the creation of micelles, we asked for help or a confirmation for details.