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<p>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 the blue chromoprotein gene is before the T7 promoter and RBS and this is a result if otherwise restrictions, ligations and transformation were successful.</p>
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<p>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 the blue chromoprotein gene is before the T7 promoter and RBS and this is a result if otherwise restrictions, ligations and transformation were successful.</p>
  
 
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<p style="margin-bottom:0; padding-bottom:10%;">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 for help or a confirmation even for a small detail.
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<p style="margin-bottom:0; padding-bottom:10%;">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 for help or a confirmation even for the smallest details.
 
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Revision as of 14:53, 13 September 2015

Practice project: Testing RBSs

Introduction

In synthetic biology it is essential to produce a enormous amounts 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. 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 for cell growth. A ribosomal binding site 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.

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 in the Laboratory section.

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 the blue chromoprotein gene is before the T7 promoter and RBS and this is a result if otherwise restrictions, ligations and transformation were successful.

Figure 1: 3A assembly. The right side shows the best case of a scenario of our attempt of the 3A assembly.

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 for help or a confirmation even for the smallest details.