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Introduction to the Lab Work
Objective
The goal of our wet lab work is validate our software prediction. There are very limited terminator efficiency data available in literatures, we have designed 100 terminators bioinformatically, and want physically measure the efficiency. By fitting our prediction with experiment results, we can adjust the parameters in our model to make it more accurate.
1, How to measure efficiency:
In figure above, we show our plasmid design to measure terminator efficiency. The terminator to be characterized is flanked by two fluorescent proteins, GFP and RFP. If the terminator is 100% efficient, then, RFP will be transcripted while GFP should not be transcripted. If the terminator is 0% efficienct, then, both RFP and GFP will be transcripted at the same time. We use flow cytometry to measure the fluorescence strength of GFP and RFP.
The terminator efficiency of a terminator is calculated as:E = 1- S/T
Here S is the fluorescence strength of GFP with terminator, and T is the fluorescence strenght of GFP without terminator, which is the baseline strength.2. Plasmid Construction
The goal of BioBrick is to standardize the form of genetic components to allow idempotent reactions where the key structural elements of a component are unchanged by the reactions. So BioBrick provide 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)
They have the same cohesive terminus so they can connect together. However, after the combination, neither XbaⅠnor SpeⅠ can recognize this sequence. According to this way can we achieve the goal of BioBricks. We can see from the picture(Figure 2) that there are 4 standard restriction enzyme cutting sites: EcoRⅠ, XbaⅠ, SpeⅠ,PstⅠ.1. Because 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 like PstⅠ,they just have 2 different base-pairs, we use PtoA-F 5'-CCACCTGACGTCTAAGAAAC-3' PtoA-R 5'-CTTAAGCGGCCGCTACTAGTA-3' These primer to mutate the vector.2. After the mutation is successful, the next step is to connect GFP & RFP to the vector. The GFP and RFP were built by PCR extension. To digest GFP, we use SpeⅠand AflⅡ. We also digest the vector with the same restriction enzyme cutting sites. During the process of base complementary pairing, GFP can be ligated into the vector with the help of T4 DNA ligase. At the same time, the recognition site of PstⅠ has been added to the vector. To digest RFP, we use NotⅠ and SpeⅠ. We also digest the vector with the same restriction enzyme cutting sites. During the process of base complementary pairing, RFP can be ligated into vector with the help of T4 DNA ligase. At the same time, the recognition site of XbaⅠ has been added to the vector. We can either connect GFP, RFP and the plasmid these 3 fragments together or connect 2 fragments together first and then connect the other one. However, when we carry out the second scheme, we should consider which one should be connected first. According to the picture, we should connect GFP first. 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. Until these steps, we have already built the vector well. 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.
