Electronic Supplement Scheme of lambda phage structure and protein components Head Tail gpA gpG gpB gpH gpC gpI gpD gpJ gpE gpK gpFⅡ gpL gpW gpM gpT gpU gpV gpZ Preparation of Lambda phage The procedure for preparing lambda phage was adapted from protocols in Molecular Cloning: A Laboratory Manual [38]. Materials ‐ media and buffer E.coli bacteria strain LE392 and lambda phage strain were purchased from Promega (Madison, WI). Sterilized Luria Bertani (LB) medium (10 g trypton, 5 g yeast extract and 10 g NaCl / liter, pH=7.5) was used for bacterial and phage cultivation, as well as for the plaque assay. Sterilized SM solution (5.8 g of NaCl, 2 g of MgSO4∙ 7H2O, 50 ml of 1 M Tris‐HCl at pH=7.5, and 5 ml of 2% Gelatin / liter) was employed as suspension buffer for lambda phage. Agar solution (7 g agar in one liter of LB medium, pH=7.5) was prepared for multiple usage throughout the procedure. Agar solution condensed in Petri dishes is referred to as bottom agar/agar plate, while agar solution stored in bottles for further use is top agar. Bacterial growth The bacteria strain LE392 was streaked on agar plate and incubated at 37 °C overnight. Then a single bacterial colony was picked from the streaked plate and incubated with 50 ml of LB medium in the presence of 0.2% maltose overnight for bacterial growth. After centrifugation of bacterial culture, the supernatant was discarded and bacterial cell pellet was resuspended with 10 mM MgSO4 to a final concentration of OD600 = 1.0 (optical density measured at 600 nm). Infecting plating bacteria /Phage plaque assay To prepare lambda phage stock and measure the concentration of the lambda phage, plating bacteria were infected with diluted lambda phage to form plaques in agar plate. First, the lambda phage (initially purchased stock or phage culture) was serially 10x diluted (10‐1, 10‐2, to 10‐8) with SM buffer. Then 100 μl of each selected diluted lambda phage serials (e.g. 10‐7, 10‐8) were gently mixed separately with 200μl of OD600 = 1.0 bacteria in small culture tubes. After incubating in a 37°C water bath for 30 min to allow the phage particles to adsorb to the bacteria, the mixture is added to 3 ml of molten top agar (47°C) and, immediately poured onto the agar plate. After overnight incubation at 37°C, transparent concentric plaques could be observed for further counting or selecting: the concentration of lambda phage can be calculated in plaque‐forming units (pfu) per ml after counting the numbers of plaques on the plate. The well isolated plaque can be selected and picked to prepare genetically homogeneous lambda phage stocks. Preparation of lambda phage stock One well isolated lambda phage plaque was picked from the plate and suspended in 1 ml of SM buffer with 50 μl of chloroform for at least 2 hours to assist phage particles in diffusing from the agar. 1/10 of suspended individual plaque solution and 200 μl of OD600 = 1.0 bacteria were used to perform the infecting plating bacteria /phage plaque assay step on 2 agar plates. After overnight incubation at 37°C, 5 ml of SM buffer was added to each lysed plate and shaken on a shaking platform at 4 °C for 4 hours. The collected SM (containing lambda phage and bacterial debris) was added with 0.1 ml of chloroform and centrifuged to remove the bacterial debris. Supernatants were collected, combined with chloroform and stored at 4 °C as lambda phage stock. Phage plaque assay was carried out to determine the concentration ((pfu ml‐1)) of this lambda phage stock, thus with proper dilution, a certain number (pfu) of lambda phage can be taken for the sequential steps. Large scale phage culture preparation 7 10 of lambda phage from phage stock were mixed with 2 ml of OD600 = 1 bacteria. After 30 min incubation at 37 °C water bath, the mixture was poured into a flask containing 500 ml of pre‐warmed LB media. Then after 9 hours of incubation with shaking rate of 275 opm, culture lysis was observed. Then 10 ml of chloroform was added to flask, and incubation continued for 20 min. Precipitating lambda phage particles The cooled lysates were treated with DNase I to a final concentration of 1 µg ml‐1 and incubated for 30 min. Then 29.2 g of NaCl was added. After settling on ice for 2 hrs, the culture was centrifuged at 11,000 g for 10 min to remove debris. 50 g solid PEG 8000 was then dissolved in the supernatants, which were kept on ice for 2 hrs. The treated culture was spun again at 11,000 g for 10 min at 4 °C and the supernatants were discarded, while phage pellets on the centrifuge bottles were re‐suspended with 10 ml of SM buffer. Equal volumes of chloroform were mixed with re‐suspended phage by gentle shaking. After centrifugation at 4000 g for 15 min, purified lambda phage, which is the lambda phage particles suspended in SM buffer, was obtained by recovering the aqueous phase. Additional Implications of this Study for Phage Technologies Phage Display Technology Phage display is a significant technology for which a foreign gene sequence(s) is inserted into the gene of the encoding phage proteins, and ultimately display an extension peptide or protein on the viral coat by expressing and amplifying in the host bacterium [48, 49]. Phage display libraries are mixtures of displayed phages carrying a different foreign gene insert. By screening phage display library with a target substance such as metal, receptor or surface, those displayed peptides or proteins that have specific affinity to the target substance will be selected out for their further applications in various areas such as drug discovery, epitope mapping, disease diagnostics and new material development. While phage display has primarily focused on lysogenic filamentous phage M13, fd and f1, there are many studies yet to be done with these and even more so with lytic lambda phage, T4 and T7 [49, 50]. For lysogenic phages, the expressed peptides/proteins need be extruded or secreted across the bacterial membranes, which may cause difficulties regarding the secretion of larger foreign peptides or proteins [49, 51, 52]. The lytic phages that are assembled in the cytoplasm of host cells and released by lysis, do not require proteins (including fusion peptides/proteins) to be translocated across the host membrane, therefore, represent advantages in ultimately displaying sizeable peptides or proteins, and even those may toxic to the host cells [49]. It has been shown that gpD and gpV in lambda phage can be employed for phage display and it is worthy to note again that these proteins are rich in metal binding sulfur. [53‐55] With the possibility of gpE, gpD, gpV interactions with the metals detected (Mn, Fe, Co, Ni, Cu and Zn), libraries might be selected or developed considering their potential interactions with phage proteins, since gpE is essential for capsid formation, while gpD and gpV have been noted as phage display proteins [45, 54, 55]. The growth methods could consider appropriate metal concentrations for displaying specific peptides or proteins on gpD or gpV. A particular area of potential benefit is biomineralization, which finds significant metal‐binding peptides or proteins serving as templates for bio‐synthesizing nanoscale materials in vitro.[20, 56] The detailed approaches including the pinning process have been described [50, 56, 57]. In general, by genetically engineering the phage coat as a presentation vehicle, numerous (e.g. ~ 109 peptides) different viral‐displayed peptides or proteins are screened with target inorganic metals to identify peptides/proteins exhibiting preferential affinity to target metals and are potentially useful for metal material bio‐synthesis [13]. So far, Ag[57], Al[58], Au[18], Co[14], Fe[58], Li[15] and Pt[17] nanowire materials have been nucleated and grown on the peptides or proteins selected from phage display libraries. The current phage vector systems engineered for preparing these materials are filamentous phages. But charged peptides or cysteine‐rich peptides that are excellent for metal‐binding might have difficulty in fusing on filamentous phages due to the limitations imposed by secretion [49, 59]. The lytic phages (λ, T4 and T7), which are free of these limitations, can be considered as alternative systems to display peptides/proteins carrying higher metal‐binding potential. This study using SEC‐ICPMS to screen metals in lambda phage samples, for ultimate metalloprotein identification, indicates phage associated metals and non‐ associated metals generate information to assist selection of preferable target metal pools for existing display libraries. Since foreign peptides/proteins can be displayed on gpV and gpD, while gpE connects to gpD trimers, if any of the lambda phage‐associated metals is chosen as target, these lambda phage proteins will compete with the displayed peptides/proteins for binding with a target metal. Also, in the pinning process, the mutant phages that contain a target metal binding peptide/protein are selected and enriched by affinity selection of a phage display library on the immobilized target so that binding phages are captured and non‐binding phages are washed away [50]. Some phages with fused non‐target metal‐binding peptides/proteins could be trapped due to the interaction between the phage proteins and immobilized target metal, thereby causing false selection of specific peptides/proteins. Hence, those lambda phage non‐ associated metals from this study (Al, K, Ca, Cr, As, Se, Ag, Cd, Cs and Hg) may serve as better choice of target metals when using lambda phage display libraries to select proper templates for target metal biomineralization.