Our team designed 15 basic parts and 3 composite parts, totaling 15 parts.
   This year, we designed BBa_K4113022 and BBa_K4113025 to increase the GABA content in longan healing tissue. If you want to see more information about them, you can click on the part number to search!

1 Basic Parts

Name	Type	Description
BBa_K4113008	Primer	DlGAD2-F
BBa_K4113011	Primer	DlGAD2-R
BBa_K4113009	Coding	DlGAD2
BBa_K4113010	Coding	DlGAD5
BBa_K4113012	Primer	U626-IDF
BBa_K4113013	Primer	DlCaM1-AtU6-1T1R
BBa_K4113014	Coding	DlCaM1
BBa_K4113015	Coding	DlCaM2
BBa_K4113016	Primer	DlGAD5-F
BBa_K4113018	Primer	DlCaM2-AtU6-1T1R
BBa_K4113019	Primer	DlGAD5-R
BBa_K4113026	Terminator	NOS terminator
BBa_K4113027	Promoter	CaMV35S Promoter
BBa_K4113020	plasmid	pCAMBIA1301
BBa_K4113021	plasmid	pCAMBIA1301SN

2 Composite parts

Name	Type	Description
BBa_K4113028	plasmid	pCAMBIA1301SN
BBa_K4113022	plasmid	pCOMBIA1301SN-DlGAD2
BBa_K4113025	plasmid	pCOMBIA1301SN-DlGAD5

Commonly used binary expression vectors for plant

2.1 Commonly used binary expression vectors for plant——pCAMBIA Vectors

plasmid Basic Skeleton
pCAMBIA1301 plasmid(Kanamycin)
pCAMBIA1301SN plasmid(Kanamycin)
pGW3(Genovo-W-3) (Source: The New Zealand Institute for Plant and Food Research Limited)

pCAMBIA1301SN-DlGAD overexpression vector

2.2 pCAMBIA1301SN-DlGAD overexpression vector

Note: LB: the left end of T-DNA region; RB: right end of T-DNA region

Left direction	name	function
	35S promoter	Marker gene promoter
	Hyg	Marker gene promoter
	35S polyA	Marker gene terminator
Right direction	LacZa	Enhanced promoter
	35S promoter	Objective gene promoter
	ERF6	Enzymatic cleavage site
	35S promoter	GUS promoter
	NOS	reporter gene
	GUS gene	marker gene
	NOS	reporter gene

BBa_K4113022
   The constructed recombinant plasmid pCOMBIA1301SN was used as a template for PCR using specific primers for the DlGAD2 gene of Longan. The results were shown in Figure 1, and the size of the PCR amplification product was about 1428 bp, which was consistent with the size of the DlGAD2 gene of Longan. To further verify the accuracy of the insertion site of DlGAD2 gene in Longan, the recombinant plasmid pCOMBIA1301- DlGAD2 was verified by double digestion using restriction endonucleases FastDigest KpnⅠ and FastDigest SalⅠ, and the digested product was imaged by electrophoresis gel, as shown in Figure 3, where the serial number ② represents the DlGAD2 after double digestion which length is 1428bp , and serial number ④ represents the band of pCOMBIA1301SN after double digestion by restriction endonucleases FastDigest KpnⅠ and FastDigest SalⅠ, indicating the correct insertion site of DlGAD2 on the vector. The combined results of PCR and double digestion showed that the DlGAD2 gene was successfully inserted into the polyclonal site of the intermediate expression vector pCOMBIA1301SN, which can be used for subsequent studies. Figure 1：PCR amplification product of DlGAD2
BBa_K4113025
   The constructed recombinant plasmid pCOMBIA1301SN was used as a template for PCR using primers specific for the DlGAD5 gene of Longan. The results were shown in Figure 2,and the size of the PCR amplification product was about 1473bp, which was consistent with the size of the DlGAD5 gene of Longan. To further verify the accuracy of the insertion site of the DlGAD5 gene in Longan, the recombinant plasmid pCOMBIA1301SN- DlGAD5 was double digested with restriction endonucleases FastDigest KpnⅠ and FastDigest PstⅠ, and the digested product was imaged by electrophoresis gel, as shown in Fig3. The serial number ① represents the band of 1473bp DlGAD5 after double digestion, and serial number ③ represents the band of pCOMBIA1301SN after double digestion with restriction endonuclease FastDigest KpnⅠ and FastDigest PstⅠ, indicating the correct insertion site of DlGAD5 on the vector. The results of combined PCR and double digestion indicated that the DlGAD5 gene was successfully inserted into the polyclonal site of the intermediate expression vector pCOMBIA1301SN and could be used for subsequent studies.
Figure 2 PCR amplification product of DlGAD5 Figure 3 Double digestion products

The pGW3 system edits the DlCaM gene

2.3 The pGW3 system edits the DlCaM gene

   When sgRNA was constructed with pGW3 (Kanamycin resistance in bacteria and Hygromycin resistance in plant) vector, the backbone fragment was 15,429 bp after digestion with BsaI, and the gum was recovered and ligated to sgRNA, and the sgRNA primers were synthesized with the addition of the vector complementary to the sticky end sequence. Using the pGW3 recombinant vector we successfully knocked out DlCaM1 and DlCaM2. Plasmid pGW3 was digested with Bsa I: Figure 4 Enzyme-digested product Figure5: pGW3-ΔDlCaM1 recombinant vector and pGW3-ΔDlCaM2 recombinant vector Agrobacterium bacteriophage PCR