As mentioned before, the key of developing a successful in vivo scaffold system is to establishing highly-specific and efficient interaction between protein and other biomolecules. Thus, the current development of genome editing technique may give us a clue by achieving those requirements spontaneously.

ZFN, TALEN and CRISPER-CAS9 are three famous genome editing techniques nowadays. Among those techniques, ZFN is now being gradually replaced by TALEN and CRISPER-CAS9, and its binding mechanism with DNA is similar to TALEN by the view of molecular biology; CRISPER-CAS9 technique is based on the specific recognition of the gRNA and the target DNA, as well as the relevantly nonspecific interaction between CAS-family protein and gRNA.

Specifically, due to the nonspecific interaction between enzyme-fused dCas9 proteins and gRNA, different engineered dCas9 proteins have to compete for gRNA, thus makes the scaffold system unpredictable and uncontrollable.

The TALEN technique is based on the direct interaction between TALE protein and the target DNA sequence. This special characteristic gave us a clue to build a scaffold system with high-specificity and efficiency.

a. As mentioned in our project description, different TALEs share a similar domain structure that enables them to bind the genome of the host cell and act as transcriptional effectors. By engineering those structures, we can build proteins that can bind with any DNA sequence that we desire.

In our project, we designed two different DNA binding motifs (DNA BMs). The sequences were chosen from Danio rerio CD154 gene in order to avoid homology with E.coli genome. Those BMs are sequenced as

BM1: 5’-GGAGGCACCGGTGG-3’

MB2: 5’-GATAAACACCTTTC-3’

Those sequences were repeated for more than 10 times in a plasmid, with different length of intervening sequence.

b. The length of DNA BMs and intervening sequences is designed based on the typical in vivo B-type DNA structure, and the structural basis for TALEs targeting DNA. Accordingly, three different scaffolds were designed to figure out the influence of the spatial orientation and hindering effect on the effectiveness of the scaffold system.

1. The first scaffold (A.K.A. SCAF1 or S1) is designed to put enzymes as close as possible, no intervening sequence is inserted between two BMs;

2. The second scaffold (A.K.A. SCAF2 or S2) is designed to avoid steric hindrance and to ensure the spatial orientation of enzymes;

3. The third scaffold (A.K.A. SCAF3 or S3) is designed to test the effect of different length of intervening sequence. The spatial orientation of enzymes is also considered.

c. Based on the scaffold we designed, we engineered three different TAL effectors, namely TALE1 (T1), TALE2 (T2) and TALE3 (T3)

TALE1 can specifically recognize the sequence of BM1: 5’-GGAGGCACCGGTGG-3’

TALE2 can specifically recognize the sequence of BM2: 5’-GATAAACACCTTTC-3’

TALE3 can specifically recognize the REVERSED sequence of BM2: 5’-CTTTCCACAAATAG-3’

d. The construction of the TAL effectors

NJU_CHINA finished the construction of our first TAL effector (TALE1) and edited the vectors provided by the kit (adding corresponding cutting sites to be specific) to meet the RFC10 bio-brick standard.

With their help, we finished the construction of the rest of the TALEs by ourselves using the Golden Gate TALEN and TAL Effector Kit 2.0 by Addgene(R). The protocol can be reached at [PDF]TAL effector construction.

Sequence of TALE1 cDNA and deduced amino acids

Sequence of TALE2 cDNA and deduced amino acids

Sequence of TALE3 cDNA and deduced amino acids

e. Designing of TALE/SCAF pair

Three pairs of TALE/SCAF were chosen for further experiments.

1. TALE1/TALE3 with SCAF1

2. TALE1/TALE2 with SCAF2

3. TALE1/TALE2 with SCAF3

The efficiency of these TALE/SCAF pairs can be determined by ChIP-PCR.

a. Split GFP

Split GFP is a technique that has been widely used in the research of protein-protein interaction. In our project, we demonstrated a prototype by fusing the Amino (or Carboxyl) Half of GFP with TALE1 (or TALE2/3).

By integrating the coding sequences of the TALE-fused proteins and the scaffold, three different plasmids could be constructed.

1. Split GFP fused with TALE1/TALE3 on SCAF1

2. Split GFP fused with TALE1/TALE2 on SCAF2

3. Split GFP fused with TALE1/TALE2 on SCAF3

This prototype is designed to test if our system can achieve our goal of compartmentation by examining if the green florescent intensity raised observably.

Fluoroskan Ascent FL by Thermo can be used to detect the fluorescence intensity.

b. IAA production

Our second prototype is designed to determine if our system can actually improve the productivity of a multi-enzymatic system.

The IAA production pathway is chosen for this purpose. The IAA producing pathway originates from Pseudomonas savastanoi contains two enzymes, AKA IAAM and IAAH. IAAM is the tryptophan-2-mono-oxygenase. This enzyme catalyzes the oxidative carboxylation of L-tryptophan to indole-3-acetamide. IAAH is the indoleacetimide hydrolase. This enzyme catalyses the hydrolysis of indoleacetamide to indoleacetate and ammonia. This pathway is relevantly easy to be built in prokaryotic cells, and its substrate (Trp) can be synthesized by host cells.

Thus, we fused the IAAM (or IAAH) with TALE2 (or TALE3). By integrating the coding sequences of the TALE-fused proteins and the scaffold, two different plasmids can be constructed.

1. IAA production pathway fused with TALE1/2 on SCAF2

2. IAA production pathway fused with TALE1/2 on SCAF3

The productivity of IAA can be determined by ELISA.