Introduction to the Lab Work

Objective

The goal of our wet lab work is to validate our software prediction. Since the terminator efficiency data available in literatures is very limited, we designed 100 terminators through the knowledge of terminator structure, and designed an experiment to measure terminator efficiency. By analyzing experiment results, we can adjust the parameters in our model to make it more accurate.

1, How to measure efficiency:

The figure above shows the plasmid designed to measure terminator efficiency. The terminator to be characterized would be flanked by gene sequences of two fluorescent proteins, GFP and RFP. By using flow cytometry, we could measure the fluorescence strength of GFP and RFP, so as to measure the terminator efficiency.

E = 1- S/T

2. Plasmid Construction

A goal of synthetic biology is to standardize the form of genetic components to prevent structural elements of components from changing with reactions. It provides a standard method of assembling genetic components using specified prefixes and suffixes. Prefixes include EcoRⅠ and XbaⅠ; suffixes include SpeⅠ and PstⅠ.

The recognition site of XbaⅠ:

(Figure 3)

The recognition site of SpeⅠ:

(Figure 4)

The same cohesive terminus enable them to connect with each other. However, after the combination, neither XbaⅠnor SpeⅠis able to recognize this sequence. Through this way we could achieve the goal of building BioBricks.

We can see from the picture(Figure 2) that there are 4 standard restriction enzyme cutting sites: EcoRⅠ, XbaⅠ, SpeⅠ,PstⅠ.

(1) As we will use pstⅠto be the restriction cutting site of terminators, if we want to ligase GFP to the plasmid, this restriction cutting site should be mutated.

The recognition site of PstⅠ:

(Figure 5)

The recognition site of AflⅡ:

(Figure 6)

Since AflⅡis similar to PstⅠ,they just have 2 different base pairs, we used these primers:

PtoA-F

5'-CCACCTGACGTCTAAGAAAC-3'

PtoA-R

5'-CTTAAGCGGCCGCTACTAGTA-3'

to mutate the vector.

(2) After the mutation succeeded, the next step is to connect sequences of GFP & RFP to the vector. These sequences were built by PCR extension.

To digest GFP sequence, we used SpeⅠand AflⅡ. We also digested the vector with the same restriction enzyme cutting sites. During the process of base complementary pairing, GFP sequence could be ligated onto the vector with the help of T4 DNA ligase. At the same time, the recognition site of PstⅠ would be added to the vector.

To digest RFP sequence, we used NotⅠ and SpeⅠ. We also digested the vector with the same restriction enzyme cutting sites. During the process of base complementary pairing, RFP sequence could be ligated onto vector with the help of T4 DNA ligase. At the same time, the recognition site of XbaⅠwould be added to the vector.

We can either connect 3 fragments: GFP, RFP sequences and the plasmid at the same time, or connect 2 fragments first and then connect the other one. However, when carrying out the second scheme, we should decide which one to be connected first. According to the picture, GFP should be the one.

Besides:

We designed these primers to amplify RFP.

R-NPS-F

5'-TATAGCGGCCGCCTTAAGTAAGTAAGAGTATACG-3'

R-NPS-R

5'-CGGAGACTAGTCTGCAGATCACATAAGTAAAGTGATAATC-3'

We designed these primers to amplify GFP.

G-SXA-F

5'-CTAGACTAGTTCTAGAGGCGGACTCACTATAGA-3'

G-SXA-R

5'-CCGACTTAAGGGATCCTATAAACGCAG-3'

(3) Since we built up the vector through the steps before, we decided to use XbaⅠ,PstⅠto digest the vector.

The terminators were built by PCR extension.

Then we can ligated terminator into the backbone and transformed into competent cells.

Characterization devices testing the performance of the terminators utilized fluorescent proteins to measure input and output and altered the arabinose transport system to control inputs. The fluorescence produced by the characterization devices were then measured using flow cytometry to calculate the termination efficiency of the terminators.

3. Fluorescence strength quantification

We used flow cytometer to measured the fluorescence strength. Laser was used to be the luminous source and then radiated perpendicular to the sample flow. Under the irradiation of laser, cells expressed fluorescent protein. We calculated the average expression level of those cells and got the average strength of GFP. We also used fluorescence microscope to take a picture of bacterial culture.

4. Agreement with theoretical prediction

According to our experiment measured terminator efficiencies and our software predicted d scores, we created a fit curve to relate all the data. The corelation coefficient is 0.8. So we have achieved a good agreement between experimental efficiency and TTEC predicted efficiency.

5. Technical Standard

Our terminator efficiency measurement protocol has been submitted to Biobrick foundation as a technical standard(BBF RFC 90).