Gibson

In order to accomplish an efficient and economical DNA fragments assembly, we adopted an enzymatic assembly method named “Gibson assembly” which is wildly used because of its brilliant effectiveness in yielding deserted DNA fragments that may up to several hundred kilobases. In our project, we elaborately modified original Gibson assembly protocol, made it both better and cheaper. And subsequently, we will summarize our practical protocol in detail.

This figure indicates the overall principle of Gibson assembly. In this figure, two DNA fragments which share the same overlapping pieces, are joined together in a one-step isothermal reaction (or precisely called One-step isothermal in vitro recombination). Because the two adjacent DNA fragments share terminal sequence overlap, when T5 exonuclease remove nucleotides from 5’ ends of double-stranded DNA, reminded single-strand overlap region can pair with each other, just like two sticky ends. After two overlapping single-strand DNA annealed, Phusion DNA polymerase fill the gaps and Taq ligase, seals the nicks (It seems not so much complicated, isn’t?  ).

The Protocol of Gibson Assembly:
The required key reagents in Gibson assembly:
T5 exonuclease (NEB, 10U/ul, 1000U )
Phusion DNA polymerase (Thermo, 100 x 50 μl)
Taq DNA ligase (NEB, 10 000U）

First, prepare DNA assembly reagent-enzyme mix (which can be stored at -20℃ until needed). The reagent-enzyme is composed of reaction buffer and enzyme which are commercial available.
And these are our reaction buffer:
5× Isothermal reaction buffer preparing protocol:

1.5 ml	1 M Tris-HCl pH 7.5 (Prepared by adding HCl into 6.05g Tris-base to adjust pH to 7.5. The total volume is 50ml)
75 ul	2 M MgCl2,        （1.9g/10ml）
30 ul	100 mM dGTP,
30 ul	100 mM dATP,
30 ul	100 mM dTTP,
30 ul	100 mM dCTP,
150 ul	1M DTT,
0.75 g	PEG-8000，
150 ul	100 mM NAD        （0.33g/5ml）
ddH2O	3 ml.

Assembly master mixture:
320 ul 5* isothermal reaction buffer,
0.64 ul of 10 U /ul T5 exonuclease,
20 ul of 2 U/ul Phusion DNA polymerase,
160 ul of 40 U /ul Taq DNA ligase,
Add water up to a final volume of 1.2 ml.

The volume of mix and DNA is 3:1. For example, 15ul mix + 5ul DNA solution.

To start a Gibson assembly, you can just simply mix the reagent-enzyme mixture with DNA fragments solution, and put the PCR tube that with the mix-DNA mixture into PCR instrument, incubate it at 50℃ as few as 15 minutes, and then the assembly of product- a recombinated DNA fragments is accomplished! By ordinary PCR procedure, we can subsequently amplify the product, for further experiment.

Measurement

To fully test our Cas-9 mediated multi-stage control system, we must establish a method that can measure a gene’s expression level preciously. We choose RFP as our report gene because its expression product can be measured easily. We can not only check whether the RFP is expressed by naked eyes, but also can determine the absolute fluorescent intensity of RFP by Multi-FunctionalXXXXXXX accurately. However, we cannot just simply adapt absolute fluorescent intensity as the RFP expressive level, because the absolute fluorescent intensity can be influenced directiy and greatly by bacterial cell density. The cell density values varies significantly among different samples, so the absolute fluorescent intensity cannot reflect the RFP expressive level truly. So here we adopted a traditional but effective trick to emit the negative affect bacterial cell density.
We define I as the relative fluorescence intensity, OD as XXXXXXXXXXX, and F as absolute fluorescence intensity. Thus, I can be formulated like this:
I = F/OD
Now, the relative fluorescence intensity won’t be influenced by bacterial density, and it obviously can reflect the RFP’s expressive level more accurate than fluorescent intensity.

OverlapPCR

Overlap extension polymerase chain reaction in gRNA coding DNA assembly
The overlap extension polymerase chain reaction (or OE-PCR, Overlap PCR), was developed in late 1980s. And until up to now, it has become a classical method for accomplishing mutations introduction and DNA molecules splicing.
In our project, OE-PCR is used in bio-brick construction---mainly in gRNA coding DNA (shortly referred as gRNA) synthesis. Synthesizing a complete, long gRNA is impracticable expensive. Nevertheless, we must synthesize several gRNA de novo. So, in order to save our budget, we ordered several short fragments which can anneal head-to-tail by short overlapping regions at their ends, and thus these fragments can cover the overall complete gRNA . By deliberately design primers, we successfully fused these DNA fragments into a complete, covalently sealed gRNA respectively by OE-PCR.
Figure 1 indicates the principle of OE-PCR, and is quite simple and plain. OE-PCR can be performed by ordinary PCR instrument. First, we separate the complete gRNA into 3 pieces, and asked ShengGong bio-technological company to synthesize these DNA pieces, and some designated primers. Each of them shares an overlapping region with its adjacent partner.

After annealing when replication occurs, the DNA piece is extended by a new sequence that is complementary to the molecule it is to be joined to. Once both DNA molecules are extended in such a manner, they are mixed and a PCR is carried out with only the primers for the far ends. The overlapping complementary sequences introduced will serve as primers and the two sequences will be fused. This method has an advantage over other gene splicing techniques in not requiring restriction sites.