Difference between revisions of "Team:BGIC-Union/Parts"

We create four Basic Biobricks regarding to T7-dCas9.
NT7-dCas9 BBa_K2371000
CT7-dCas9 BBa_K2371001
NT7 BBa_K2371002
CT7 BBa_K2371003

Description:

The first two Biobricks are the two split T7 polymerase sequence we adopted. We provide them for other team to leverage them through other methods. We choose the split site suggested by the team of Tiyun Han, Quan Chen and Haiyan Liu. [7]
The following two Biobricks are the sequence of T7 polymerase connected to dCas9 protein. The linker between split T7 polymerase and dCas9 is GGGGSGGGGS adopted by Peking University. A 6 X His-tag is added on C terminus of dCas9 in order to purify it.

We create seven composite Biobricks regarding to sgRNA.
We created a Biobrick for each of sgRNA and insert T7 promotor and T7 terminator before and after the sequence respectively in order to utilize them to transcribe sgRNA in vitro. The sequences are synthesize through Oligo DNA bridging designed by DNAworks. The detail of synthesis method is in the experiment.

Figure 1. The plasmid of sgRNA generator. The sgRNA generator Biobrick is composed of a T7 promotor, coding sequence for sgRNA and a T7 terminator.

sgRNA generator for EML4-ALK Variant A 23 BBa_K2371004
sgRNA generator for EML4-ALK Variant A 33 BBa_K2371005
sgRNA generator for EML4-ALK Variant A 83 BBa_K2371006
sgRNA generator for EML4-ALK Variant A 93 BBa_K2371007
sgRNA generator for EML4-ALK Variant B 70 BBa_K2371008
sgRNA generator for EML4-ALK Variant B 126 BBa_K2371009
sgRNA generator for EML4-ALK Variant B 147 BBa_K2371010

We create two supplemental composite biobricks for expression of GFP and RFP in cell free system.
pT7-eforRed BBa_K2371011
pT7-amilGFP BBa_K2371012
These two biobricks are composed of a T7 promotor with a coding sequence of GFP or RFP.

Figure 2. The plasmid of pT7-RFP. pT7-RFP is composed of a T7 promotor, an RBS and the coding sequence of RFP. This plasmid is used to act as report gene in our report system.

Figure 3. The plasmid of pT7-amliGFP. pt7-amliGFP is composed of a T7 promotor, an RBS and the coding sequence of RFP. This plasmid is used to act as report gene in our report system.

These two biobricks are supposed to cooperate with T7 PC report system in cell free system. The user need to conduct a PCR to linearize the part before reacting with T7 report system. When the split T7 polymerase reassemble, it will start transcription of GFP and RFP from these two plasmids.
We conducted both PCR check(VF and VR) and enzyme check(XbaI and SpeI)


Figure 4.PCR check of eforRed and amliGFP. The expected result of eforRed should be around 1000bp. The insufficient specificity of primers produce another band but the band around 1000bp can present the success of construction. The expected result of amliGFP should be around 1000bp. The control is J23119 biobrick.

Figure 5.RD check of eforRed and amliGFP. XbaI and SpeI were used. The expected length of both amliGFP and eforRed should be around 750bp.