Introduction

In synthetic biology it is essential to produce an enormous amount of 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. The more product is formed when host cells are big and physically stable.

According to Ceroni et al. [1], strong ribosomal binding sites (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 to cell growth. A ribosomal binding site is a location in an mRNA which a ribosome recognizes and binds to initiating translation. The RBSs is defined by efficiency which they bind to ribosomes. Thus the strong RBSs binds more efficiently than the medium or the weak RBSs.

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 a protein yield. And we also could get some practice of lab procedures.

Our goal was to create 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.

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 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 more blue the samples are the more protein had to been translated.

The detailed descriptions of our procedures and methods can be found in the Laboratory section.

Results

Our spectrometer 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 why it did not work: our 3A assembling was flawed. As shown in Figure 1. the main problem is that in the assembled plasmids the blue chromoprotein gene is before the T7 promoter and RBS and this is a result if otherwise restrictions, ligations and transformation were successful.

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 even once went to back to check our restriction plans with instructors or even within ourselves, 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 help or a confirmation even for a small detail.