Difference between revisions of "Team:HUST-China/Description"

Description

In our project of Euk.cement, we employ Yarrowia lipolytica JMY1212 as our chassis and generate three circuits respectively using expressing vector pRMH120 and JMP62 as backbone to secrete viscous protein and express its supporting and control matters aiming to consolidate sands or soil particles more flexibly. Next we will show all the details--

(Figure 1： The complete circuit)

Our design are briefly divided into three parts: light control system,flocculating system and supporting system. The latter two are both under control of the former one with the same upstream promoter Anb1. Fusion protein CRY2-BD and CIB1-AD are respectively expressed by constitutive promoters pTEF and pADH1. CRY2-BD has high affinity to bind upstream of pGal1 and CIB1-AD when induced by blue light (even nature light) can activate pGal1 owing to the combination between CIB1 and CRY2’s special structures thus inducing the downstream gene of pGal1.Then expressed ROX1 protein can inhibit the promoter Anb1. So our engineered circuit is turned off when exposed to light. On the contrary,in darkness,the structural-reversed CRY2 will dissociate with CIB1. Without CRY2-BD’s target function,AD is hard to combine and active pGal1 in the complex environment of nucleus,so the downstream ROX1 is no longer transcribed. With the degradation of translated ROX1 protein,pAnb1 start to express the downstream gene as the supporting matter and flocculating agent :LIP2 pro-Si-tag-YLcwp3 and LIP2-pro-Mcfp-3 fusion proteins(shown in Fig 1).

Darkness induction system

(Figure 2： Darkness induction system)

The first one is the control system. Each system should apply a kind of trigger without out any doubt. While a number of inducible systems are available for controlling protein expression in yeast, almost none of them make sense for us, as our project’s practical applications are currently considered to exist mainly in marine environment, which makes the amount of chemical inducers to be used a huge burden. So we give up several common promoters including those naturally responsive to molecules such as galactose, methionine, and copper, or engineered regulatory systems that respond to orthogonal molecules such as estrogen (Gal-ER-VP16)^[1] or doxycycline (Tet-OFF)^[2] . In fact, some of them are really robust and well designed pathways. However, we find a better method instead, a darkness induction system based on yeast two-hybrid principles. Our original idea comes from dimers that allow inducible control of protein-protein interactions, which are powerful tools for manipulating biological processes. On this basis, we successfully develop a split transcription factor that is reconstituted by light-dependent protein–protein interactions as in yeast two-hybrid systems. Compared to those chemical-genetic tools, the light control system not only meets our demand, but also possesses many other advantages. Despite the general success of chemical inducers, the potential problems, such as toxic, unintended, or pleiotropic effects of these treatments can impose limitations on their use in sensitive chassis or environment. Also, light control is far more fast, precise, invasive and easy. It even allows exquisite dose-dependent control over protein levels, as light can be delivered for defined amounts of time and be instantly removed.

Yeast two hybrid principle

(Fig3: yeast two-hybrid principles)

To regulate DNA transcription by light, the system is based on a two-hybrid interaction in which a light-mediated protein interaction brings together two halves (a binding domain and an activation domain) of a split transcription factor of pGal. One of the light-mediated protein is Arabidopsis CIB1, a basic helix-loop-helix (bHLH) protein, and another is cryptochrome 2 (CRY2), a blue light-absorbing photosensor that binds CIB1 in its photoexcited state^[3]. When applying the two-hybrid system to light control switch,we generate a couple of fusing protein with each half containing an light-mediated part and transcription-facter-related part. If we remove the stimulation of blue light, dark reversion of CRY2 will dissociate the interaction with CIB1 and halt Gal4-dependent transcription. These modules require no exogenous chromophore, and are reversible within minutes thus triggering protein translocation on a sub-second time scale, and even allow potential use in vivo in whole organisms (shown in Fig2).

(Figure 4： Light control)

In the circuit, the two halves, CIB1 and CRY2 are fused separately with activation domain (AD) of Gal4 and DNA binding domain (BD) of Gal4,the fusion of which are respectively expressed by constitutive promoters pADH1 and pTEF. Both of them are essential to yeast genetics as the trigger of transcription requires their attachment to the promoter sequence at the same time. Commonly, it is almost impossible because they are too far from each other. But when exposed to blue light, such interaction of CIB1 and CRY2 brings together the split Gal1(similar to Gal4) transcription factor to control DNA transcription. As BD combines to the upstream activation sequence (UAS) and AD is successfully targeted to activate the promoter, downstream gene are able to be transcribed and translated, following the principle of yeast two-hybrid(shown in Fig3).

Logic reverse control

(Figure 5： Logic reverse control)

Then a couple of promoter Panb1 and it’s inhibitory protein ROX1 ^[4] are set afterwards(shown in Fig4). Actually, a light-activated promoter as we described before is still one step away from our needs. Since the applications of this project are expected to be put into marine environment, it is highly possible that the marine yeasts are unable to get to sunlight in most of the blue water region. Based on the above,we generate a logic reverse control. After introducing Panb1 and ROX1, the system’s function is exquisitely switched: in the condition of light ROX1 in the downstream of pGal1 are expressed to inhibit expressing our functional protein. Only total darkness can remove the the inhibition allowing the expression of target protein in abysmal sea of all time.

Supporting system

Next, to preserve the kit’s function, we place a binding protein Si-tag right after Panb1. It’s not for direct consolidation, but to target marine yeasts to the surface of sands. Considering those rough waves may occur occasionally, a guarantee for the yeasts not to flow away is really necessary, which equals to immobilize them on silicon. When fused with a anchor protein YLcwp3 at the C terminator and signal peptide LIP2 prepro at the N terminator the Si-tag can be displayed on the surface of the cell wall linking the yeasts’ fate to sands thoroughly.

(Figure 6： Supporting system：displaying Si-tag on the cell wall of Y.L)

Si-tag

Si-tag is 50S ribosomal protein L2 in the genome of E.coli^[5,6] . It has been extracted from several bacterial strains and proved to have a silica-binding property. It is demonstrated that an L2-protein fusion binds to silica surfaces 20- to 100-fold more strongly than poly-Arg-tagged proteins, showing its strong superiority in this field. The Si-tag consists of three domains. We are already aware that domain 1&3 binds highly stronger than domain2. Based on this, we do a deeper dig to see how the combinations of different domain work. Indeed, we find it meaningful as a kit that fits in several circumstances. This varying levels of viscosity is just what we want a kit to be to function in flexibly. The separate ones, domain 1,2,3, and combinations like 1&2, 1&3, 2&3, 1&2&3, are all tested. Hoping a linker could bring better flexibility between two domains, we put a short one between domain 1&3, creating the 1&l&3 Si-tag.

Anchor protein

Ylcwp3 is the covalently bound GPI-anchored cell wall protein of Yarrowia lipolytica. The protein allows the target to be covalently bound to β-1,6-glucan of the cell wall. The nucleotide sequence encoding 110 amino acids of the Ylcwp3 C terminus has been widely utilized for the construction of the cell surface display vector in Y. Lipolytica. The observed high activity level of the immobilized lipase LIP2 on the surface clearly shows the efficiency of the surface display system^[7,9]. Therefore we adopt YLcwp3 as an anchor to target the interest protein on the surface of the cell wall.

Signal peptide

Yarrowia.lipolytica naturally secretes lipases LIP2. The signal peptide LIP2 prepro we employ is the modified prepro of Y.lipolytica LIP2 with the sequence consisting 13 aa pre, 4 XA or XP dipeptides, 10 aa pro and KR cleavage site. It can leads the co-translational translocation of heterologous protein Mcfp-3 to be secreted out of the cell thus achieving its function,during the progress of which the peptide will be deleted in the Golgi apparatus to eliminate the interference to the secreting protein^[8,9] .

Flocculating system

(Figure 7 Flocculating system：secreting Mcfp-3 among sand)

Followed is flocculating agent secretion circuit which briefly consists of the promoter anb1, LIP2 prepro signal peptide, Mcfp-3 sticky protein and its terminator yADH1. Mcfp-3 in the substrate functioning as the main flocculating matter has a high affinity to silica and other solid surface. So the secreting protein can cross link sand particles together making flocculation.

Flocculating agent

Mcfp-3 is foot protein secreted from Mytilus californianus^[10] . The protein is of significance to the formation of byssus to help mussels permanently or temporarily tether to the surface of solid surface of reef or ship-body. This coherent substance shows an excellent adhesion performance with no substitute under water for sustaining the repeating wash of waves and having no toxicity. The properties are owing to the amino acid content which are successively DOPA (28mol％),glycine,lysine and asparagine. The presence of DOPA in the foot protein suggests a role in protein-protein and protein-substrate(glass eg.) interactions related to the formation of adhesive plaques. The electron rich nature of DOPA and its susceptibility to oxidation can lead to the formation of o-quinone with subsequent tanning or sclerotization to form a cross-linked and stable products. Further,it is noteworthy that the protein shows an intrinsic ability of self-assemble into stable,higher molecular weight forms that may further enhance our consolidation function^[11] .

Host strain

We adopt a marine yeast Yarrowia lipolytca JMY1212 as our chassis ^[12] for the specific application this year--building artificial reef. Our host is generally regarded as safe microorganism and has an abroad application prospect in industries of foods and medicine. Researches show that Y.lipolytica is widely found in marine environment and exists stably in the ocean and healthy marine fishes’ intestine. It is able to make use of Glucose,ethanol,acetate or other hydrophobic substrate(alkanes,fatty acid and lipid ) as carbon source with fast growing capacity and adaptability for high-density fermentation. Moreover in the expression system of Y.lipolytica, transformed plasmids can integrate into multiple site and they are all genetically stable in the genome. And the signal peptide XPR2 pre,LIP2 prepro and so on are all demonstrated to own high efficiency of secreting protein.So it is obvious Y.lipolytica is a powerful host to express and secrete complex recombinant protein. Based on the above reasons,Yarrowia lipolytica are regarded as ideal strain for our marine application of micro-consolidation

Improvement

balabalabalabalabalabalabalabalabalabalabalabalabalabalaba labalabalabalabalabalabown high efficiency of secreting protein.So it is obvious Y.lipolytica is a powerful host to express and secrete complex recombinant protein. Based on the above reasons,Yarrowia lipolytica are regarded as ideal strain for our marine application of micro-solidation