expression solutions VectorGPS(R) Expression vectors Electra cloning system GeneGPS optimization Gene synthesis & custom cloning expression Expression of active protein at high levels requires selecting the best coding sequence for a gene and the best vector from which to express it. ATUM’s empirically-derived codon optimization ensures that your gene is designed for efficient translation in your chosen host. Our expression vectors combine control elements that work well together for mammalian, bacterial or yeast systems. For especially challenging proteins or new host systems, we can develop customized solutions. We can even test the expression or produce the protein for you.

ATUM has developed a variety of expression vectors, using Design-of- Experiment techniques to test interactions between control elements, secretion signals and functional tags. Vector elements also interact with the gene being expressed, so there is no one vector that will work for all proteins. We have selected the vectors in our catalog to maximize the chances that at least one will have the expression properties you need. contents Mammalian Expression: Vectors and Services 2-11 Transient, stable, Leap-In transposase and lentivirus vectors for high protein expression. Bacterial Expression Vectors 12-13 High protein expression, with a choice of promoters, markers, tags, secretion signals and more. Yeast Expression Vectors 14-15 High protein expression, with a choice of promoters, markers, tags, secretion signals and more. Electra Vector System(R) 16 One tube, 5 minutes, any ORF. Scarless cloning in bacterial, mammalian and yeast systems. GeneGPS(R) Optimization 17 One tube, 5 minutes, any ORF. Scarless cloning in bacterial, mammalian and yeast systems. Expression Services 18 Gene synthesis, Custom Cloning, LSPs, and Expression Test. VectorGPS(R) 19 Discover the vector that works best for your research. mammalian expression: vectors & services

ATUM offers vectors for transient and stable 70 mammalian protein expression. Vectors shown here are available from the ATUM catalog as easy 60 to clone Electra kits. Any synthetic gene made by pD2610-v1

ATUM can also be cloned into these vectors. 50 pD2610-v12

pD2610-v26 40 pD2600 Transient Expression Vectors pD2610-v10 pD2610-v14 We offer around 30 different transient vector 30 configurations, which we identified by testing over pD2610-v13 pD2610-v9 pD2610-v3 250 different combinations of promoters, introns, 20 pD2610-v4 pD2610-v23 pcDNA3.4 pTT5 pD2610-v27 polyadenylation sequences and viral amplifiers in pD2610-v2 pD2610-v5 pD2610-v28 HEK and CHO cell lines. pD2610-v6 10 pD2610-v11 pD2610-v29 pD609 pD2610-v17

HEK293 Expression (Fluorescence X1000) pD2610-v19 pD2610-v16 Intron ORF pD2610-v8 pD2610-v20pD2610-v18 0 pD2610-v7 pD2610-v15 0 10 20 30 40 50 CHO-K1 Expression (Fluorescence X1000) polyA Globin BGH... Promoter CMV 120 EF1a GAPDH...

pD2610-v5 Enhancer pD2600 100 CMV EF1a pD2610-v2 SV40 Synthetic... pD2610-v1 80 pcDNA3.4 pD2610-v14 pD2610-v13 SV40 ori EBV oriP pD2610-v12 pD2610-v23 SV40 large T-Ag pTT5 pD2610-v2p6D2610-v4 pD2610-v10 Bacterial resistance marker EBNA... 60 pD2610-v9

Ori pD2610-v3 pD2610-v29 The reason we have so many vectors is that even 40 pD2610-v27 similar cell lines seem to have different control pD2610-v11 Expression (Fluorescence X1000) pD2610-v30 element preferences, as is shown in the graphs TM 20 pD2610-v17 below. Added to this, differences in desired pD2610-v15 pD2610-v6 duration of culture and the localization of the Expi293 0 pD2610-v28 protein being produced (intracellular, membrane- 0 10 20 30 40 50 60 70 bound or secreted) make it impossible for a single Expi-CHOTM Expression (Fluorescence X1000) vector to meet all needs.

DasherGFP expression compared in transiently transfected CHO and HEK293 cells. Cells were harvested from independent triplicate transfections and DasherGFP fluorescence was measured 72 hours post-transfection.

2 www.atum.bio MAMMALIAN EXPRESSION

pD2600 Transient Expression Vectors 500 Rituxan 23LN Vectibix Herceptin 450 MM121V Herceptin (ATUM codon optimized)

l 400 /m

µg 350 n i c.

on 300 c dy 250 bo ti

An 200 - on

ssi 150 e pr

Ex 100 K

HE 50

0 1 7 2 5 4 9 6 3 8 10-v 10-v 10-v 10-v 10-v 10-v 10-v 10-v 10-v11 10-v 10-v17 10-v12 10-v15 10-v14 10-v16 10-v19 10-v13 10-v10 10-v18 10-v20

-50 10-v13d pD26 pD26 pD26 pD26 pD26 pD26 pD26 pD26 pD26 pD26 pD26 pD26 pD26 pD26 pD26 pD26 pD26 pD26 pD26 pD26 pD26

Antibody production in transiently transfected HEK293 and Cells were transfected at 70-80% confluency using Fugene; CHO cells. ATUM dual expression vector configurations transfections were carried out in triplicate in HEK293 and (single construct) enable co-expression of antibody’s heavy CHO cells. Antibody expression was measured by heavy and light chains by coupling expression through an IRES. chain quantitation by ELISA, 6 days post-transfection.

We express hundreds of proteins every week in HEK and CHO, so if you need help in choosing a vector for your protein, please call us! Or let us make the protein for you.

3 transient expression services

Protein Expression Whether you need milligrams of thousands of Our versatile platform produces proteins including proteins as part of a drug candidate screening antibodies, Fabs and Fc fusions at 1ml - 20l scales. process, or grams of a few proteins for in depth Delivery times are accelerated because sequence characterization of your best leads, we can help. and vector optimization, synthesis & cloning, expression and purification are all under one roof. An automated, bar-coded, time-stamped process can screen thousands of expressed proteins in parallel. For large screening projects, it can be worth spending time optimizing the components. This can include:

⬤⬤Optimizing codon bias and eliminating potentially problematic cryptic splice sites from DNA sequences.

⬤⬤Selecting efficient signal sequences for secreted proteins.

⬤⬤Identifying the best vector.

⬤⬤Balancing chain ratios for antibodies or other proteins with multiple polypeptides.

4 www.atum.bio MAMMALIAN EXPRESSION

The graph shows expression 250 of twelve representative murine antibody sequences 200 for a screening project. The genes were cloned into 5 different vectors, transfected 150 into HEK cells and harvested

5 days later. Based on this Titer in mg/l 100 data we selected vector #2 for further process 50 development.

0 1 2 3 4 5 Vectors After optimizing secretion 250 signals and constant region coding region as well as the l vector sequence, we were 200 able to exceed the target productivity of 75 mg/l 150 in >95% of the expressed antibodies. 100 Antibody Yield in mg/ 50

0 1 1 1 7 5 9 3 3 9 61 13 97 37 73 25 49 12 85 18 15 14 16 19 13 10 Constructs

Proteins are delivered with a rigorous QC package. ⬤⬤ Protein concentration (A280) ⬤⬤ Molecular weight (reducing and non-reducing gel electrophoresis) ⬤⬤ Aggregation (size exclusion chromatography)

And optionally: ⬤⬤ Endotoxin (Charles River test) ⬤⬤ Glycan analysis (mass spectroscopy) ⬤⬤ Binding affinity (Surface plasmon resonance with the Octet)

5 pD2500 & pD3600 stable vectors

ATUM offers two stable vector series: the pD2500s and pD3600s, which share a similar structure. The pD2500s are compatible with one of our Leap-In transposases (LPN-1), but they do not require Marker promoter Mammalian marker Promoter 1 a transposase for good levels of expression.

ORF1 signal The pD3600s are compatible with both Marker polyA of our Leap-In transposases (LPN-1 ORF1 and LPN-2), and we recommend that ORF1 poly A they only be used with a transposase. Insulator intervening sequence This is because we have deliberately attenuated the selectable markers in Leap-In Transposon the pD3600s, so multiple integration P kanamycin events are required for cell viability Promoter 2 under selection. The pD3600s are M_Kanamycin-r_* therefore capable of higher expression yields. ORF2 signal Bacterial Origin ORF2 The pD2500 and pD3600 vectors are Transposon right border ORF2 polyA constructed in a modular way that allows Insulator easy modification of component elements. Versions are available that allow expression of up to four open reading frames in addition to the selectable marker.

Our stable vectors are built from several sets of mammalian functional elements:

⬤⬤ Metabolic selectable markers ⬤⬤ RNA processing elements Glutamine synthase (GS), dihydrofolate Introns, post-transcriptional response reductase (DHFR) elements

⬤⬤ Drug-resistance markers ⬤⬤ Insulators puromycin, blasticidin, hygromycin, Flanking the construct, and isolating neomycin, zeocin transcriptional units from each other

⬤⬤ Promoters EF1a, CMV (murine and human), hybrids

The total number of possible combinations of these is very large, and we do not have all of these built. However construction is modular, so it is easy for us to create custom combinations if we don’t have the combination you want.

6 www.atum.bio MAMMALIAN EXPRESSION

One Vector, Two (or more) Open Reading Frames Co-transfection of separate vectors containing chain to obtain high titers. If genes encoding the genes for heavy and light chains is a common two chains are incorporated into the production method for producing antibody from transiently host genome independently, the number of transfected cells. This approach is less desirable gene copies integrated, and integration position for stable cell line generation. The production of effects, will influence the relative expression of the properly assembled antibody requires sufficient two chains. This results in a higher downstream light chain to help the heavy chain fold. However screening burden during the cell line development a great excess of light chain diverts cellular process. resources away from producing enough heavy

first second chain heavy gene ID first promoter polyA promoter ratio productivity chain 269998 EF1a-Hs a A 2.96 24,164 6,126 269986 EF1a-Hs a B 2.86 24,442 6,339 269996 EF1a-Hs a C 2.53 30,799 8,608 270003 EF1a-Hs b D 1.97 24,182 7,846 270005 EF1a-Hs c C 1.81 33,991 12,129 270000 EF1a-Hs d E 1.78 23,058 8,037 269984 EF1a-Hs b C 1.17 25,303 11,699 269976 EF1a-Hs d F 1.10 32,845 15,887 270006 EF1a-Hs e B 1.01 30,603 15,548

Genes encoding DasherGFP and CayenneRFP were cloned to calculate the relative expression of the two proteins (in into pD3641h in 9 different dual-promoter configurations arbitrary units). The total fluorescent protein expression is as shown above. Constructs were transfected with LPN-1 shown in the “productivity” column. Expression of the lower transposase mRNA into CHO-K1 GS KO cells, and selected expressing proteins is shown in the “heavy chain” column. in media without glutamine. After recovery, DasherGFP The ratio of expression of the two proteins is shown in the and CayenneRFP fluorescence were measured and used “chain ratio” column.

A preferable method is to express both genes is influenced by the 3´ sequences of the first gene, from the same construct. In this case regulatory as well as the promoter sequences driving the elements can be chosen to produce a desired second. expression ratio between the two chains. The table above shows 9 different construct configurations. Similar combinations of control elements can be The total productivity differs by less than 30% for used to create constructs carrying 3 or 4 open any of the constructs, but ratios of between 1:1 reading frames, enabling expression of all of the and 3:1 can be achieved. We find that this ratio chains for a bispecific at controlled ratios.

7 Leap-In Transposase(R)

The main limitation in creating cell lines from are integrated, sometimes with additional multi-ORF constructs is what happens to the rearrangements and concatemerization. This can DNA when it gets into the cell. Typically DNA undo all of the benefits of providing the genes in a is randomly fragmented and those fragments single construct.

Designed Construct

ITR HC LC GS ITR

+ Transposase - Transposase multiple independent integrations truncations of expression cassette of complete expression cassette plus random concatemerization and scrambling

ITR HC LC GS ITR HC LC GS

ITR HC LC GS ITR HC GS

ITR HC LC GS ITR HC

ITR HC LC GS ITR HC LC

ITR HC LC GS ITR LC GS

ITR HC LC GS ITR GS

ITR HC LC GS ITR HC LC GS LC GS

Structural integrity of the complete expression cassette structural integrity of the expression cassette is compromised is maintained in the presence of transposase as shown in as seen by truncations plus random concatemerization and the left panel. In the absence of transposase (right panel), scrambling of the expression cassette.

Transposases restore the benefit of carrying all genes on a single construct. Because the Leap-In transposases integrate the entire transposon into the expression host genome, they maintain the structural integrity of the construct, linkage of all genes to the selectable marker, and the desired expression balance.

8 www.atum.bio MAMMALIAN EXPRESSION stable cell line development services

ATUM’s Cell Line Development Services combine ATUM uses two well characterized mammalian our Stable Vectors and Leap-In transposase cell lines for bioproduction: technology with our other capabilities all housed ⬤⬤ CHO K1 GS KO cell line from Horizon Discovery under the same roof in Newark, CA. We use our codon optimization algorithms, secretion signal ⬤⬤ DG44 from the lab of Dr. Lawrence Chasin toolbox and customized vector configurations to (Columbia Univ), adapted to serum-free generate high productivity CHO-K1 cell lines. suspension

step 1 step 2 Coding sequence optimization Vector selection Expression of proteins in mammalian cells may ATUM typically uses pD3600 vectors with be limited by codon usage. The effectiveness glutamine synthase (GS), dihydrofolate reductase of different secretion signals can also be highly (DHFR) or puromycin selectable markers. A protein-dependent. If expression of your gene series of selectable marker expression levels appears limited, we recommend testing a re- are available. Lower levels of selectable marker coded version of the open reading frame, and/or a expression produce a more stringent selection, different signal sequence. which in turn results in higher expression of client proteins. 160 ATUM typically clones client sequences into several different vectors to obtain maximal )

l 140

/ expression levels. Our stable expression vectors g

m 120 have combinations of promoters for expressing (

r two, three or four open reading frames at controlled e t i 100 ratios. In some cases it may not be known what T

b 80 the desired expression ratios should be. In these a

m cases ATUM will create several different versions,

u 60 z and use pool productivity to guide us to the u t s 40 optimal configuration. a r T 20 0 0 20 40 60 80 100 Rituxan Titer (mg/l) Expression of two different antibodies (trastuzumab and rituxan shown, N=2) was tested using 24 different heavy and light chain signal sequence combinations. Each blue circle represents a combination of two signal sequences, one for each antibody chain. Signals that work well for one antibody may be mediocre or extremely poor for another. Comparable variability is seen in the performance of a signal in directing secretion of a light versus a heavy chain (not shown here). 9 step 3 Stable pool generation Plasmid DNA is co-transfected with transposase silencing after genomic integration. Multiple copies mRNA. Transposons preferentially integrate of a transposon are independently integrated into in transcriptionally active chromatin. ATUM’s the genome of a single cell, increasing expression transposons are flanked by insulators to reduce levels.

legend

Transposon Recognition Site + Transposase Inverted Terminal Repeat Expression Construct

Transposase protein

Transfect

Multiple integrations by cut & paste

Nucleus Nucleus

Cell Cell Chromosome Chromosome

Examples of stable pool productivity Cells are grown under selective conditions to produce stable pools. These are ranked by productivity and product quality. At this point material can be generated from the stable pools and single clone isolation is initiated.

GS gene MSX volumetric specific gene ID HC promoter promoter (mM) productivity day productivity 269962 IgG1-Hs h dual EF1a 15 2.1 g/l 15 19 pcd 269963 IgG1-Hs ht dual EF1a 0 4.2 g/l 18 42 pcd 269963 IgG1-Hs ht dual EF1a 0 3.6 g/l 18 29 pcd 269963 IgG1-Hs ht dual EF1a 0 3.3 g/l 18 29 pcd 269964 IgG1-Hs hx dual EF1a 5 3.3 g/l 18 19.9 pcd

CHO K1 GS KO cell line from Horizon Discovery (catalog HD- Stable pools were established and productivity measured in BIOP3) was co-transfected with ATUM’s transposase and non-optimized small scale shake flask or deep-well cultures. transposons with different vector and antibody combinations.

10 www.atum.bio MAMMALIAN EXPRESSION

step 4 step 5 Stability testing Isolation of monoclonal lines & research cell banking Single cells are isolated from selected pools using Genetic stability is assessed by demonstrating a FACS-based cell printer. Monoclonality is also consistent productivity and growth rate over 60 demonstrated and documented using a Molecular generations and by genomic analysis (Southern Devices CloneSelect Imager. Clones are ranked blotting, copy number and sequencing). by productivity and product quality. Product from Research cell banks (~30 vials) are established the best pools or clones can be provided to client from the top performing 1-3 clones. Cell banks are for additional function-specific analyses. released after testing for sterility, mycoplasma, transgene integrity by Southern blot and cDNA Standard clone ranking analysis sequence analysis.

⬤⬤ Productivity Octet and ELISA

⬤⬤ Binding affinity Octet

⬤⬤ Molecular weight Reduced and non-reduced gels Mass spectroscopy

⬤⬤ Macromolecular structures / aggregation SEC-HPLC

⬤⬤ Total glycan analysis Mass spectroscopy

⬤⬤ Thermal stability Tm analysis

11 bacterial expression vectors

ATUM offers a variety of vectors for bacterial either individually or in functionally related panels. protein expression. All of these vectors are Any synthetic gene made by ATUM can also be available from our catalog in our Electra format, cloned into these vectors at no extra cost. Promoters All of our promoter systems can yield high lev- the level of individual cells, so is the best choice els of soluble active protein, but their differences for periplasmic or secreted expression. The phoA can make one more suitable than the others for promoter is the cheapest to induce: it is repressed particular tasks. The IPTG-inducible T7 system in the presence of inorganic phosphate, and turns is familiar to most people, but requires a special on strongly once the phosphate is used up. Finally, cell strain, unlike the comparable IPTG-inducible we have a super-tightly repressed hybrid promoter T5 promoter which will work in any E. coli strain. that requires induction with IPTG and arabinose. The rhamnose promoter controls expression at

features T5 T7 Rhamnose PhoA Arabinose

expression Inducible Inducible Inducible and Titratable Inducible Inducible and Titratable

expression RBS choice RBS choice [Rha] and RBS choice Strong RBS [Ara] and tuning [IPTG]

localization Intracellular Intracellular Intracellular or Secreted Intracellular Intracellular or Secreted

host Any E. coli BL21(DE3) or Any E. coli or Any E. coli BL21 or DH5α T7 Express Gram-negative bacteria

350 Phi29 300

l DasherGFP 250 200 150

Yield in µg/m 100 50 0 promoter T5 T5 T7 T7 Rha Rha phoA Ara

copy low high low high low high high low

Protein expression from ATUM inducible bacterial number origins. Expression of phi29 (blue bars, ~61 kD, not promoters. Two different proteins were expressed from five tested in P_ara) and DasherGFP (green bars, ~26 kD) were different inducible promoters on plasmids with different copy measured by densitometry of stained acrylamide gels.

12 www.atum.bio BACTERIAL EXPRESSION

Electra right Terminator Secretion Signals P resistance marker ORF The effectiveness of a secretion signal depends upon the protein to be secreted. Our panel of high copy rhamnose-tunable secretion vectors

Tag Bacterial resitance enables you to quickly identify the right leader and MBP Amp Fh8 Chlor induction conditions for your protein. GST... Bacterial Expression Gen Kan Vector Zeo Secretion signal mal 80 MBP Alk. Phosphatase Cutinase ompC pelB... 70 RBS Strong 60 Medium Promoter Weak T5, T7 Rhamnose Ori 50 Term High Arabinose Medium, Low PhoA 40

30 Solubility Tags 20 Proteins that initially express as inactive and 10

insoluble in inclusion bodies can often be made Soluble processed (Secreted) protein in µg/ml active and soluble by fusing them to a solubility 0 l II p X B lB lB rT pT pA oA bA pC gIII mB ST ma to tol EO pe

tag. Different proteins respond to different tags. mA ds ph Mg la om om om Our tags are available in combination with T5, T7 M and rhamnose promoters. Different proteins prefer different secretion signals. Periplasmic expression of MBP (yellow circles), Cutinase 1.2 gene A gene B gene C (green circles) and Alkaline phosphatase (blue circles) from rhamnose vectors with different secretion signals. 1 Total cellular protein levels are represented as circle diameter. Soluble protein in the periplasm is shown on the y-axis. Protein was quantified by densitometry of stained 0.8 acrylamide gels.

0.6

Percent soluble 0.4

0.2

0 MBP GST PpiB Fh8 His Different proteins respond to different solubility tags. Three proteins (blue, green and yellow circles) were expressed fused to different N-terminal tags under control of the IPTG- inducible T5 promoter. An N-terminal His fusion is shown as a control. Total expressed protein levels are represented as circle diameter. Soluble protein is shown on the y-axis. Protein was quantified by densitometry of stained acrylamide gels. 13 yeast expression vectors

ORF Electra right ATUM offers a variety of vectors for yeast Secretion signal Alpha factor Term protein expression. All of these vectors are Invertase P_marker Lysozyme... available from our catalog in our Electra

Tag format, either individually or in functionally His HA... related panels. Any synthetic gene made RBS Selection marker by ATUM can also be cloned into these Strong Zeo Gen vectors at no extra cost. His3 Ura3...

Yeast Expression Term Vector

Bacterial marker Amp Zeo Chlor Kan Promoter AOX GAP ADH GAL1... Ori High

features Pichia S. cerevisiae

Inducible (AOX1) or Inducible (GAL1) or expression Constitutive (GAP) Constitutive (ADH, GPD, TEF)

Yeast self-replicating (2m) or propagation Targeted genomic insertion Targeted integration

localization Cytoplasmic or Secreted Cytoplasmic or Secreted

integration protocol Easy, requires linearization Easy, some require linearization

expression markers Antibiotic (Zeo) Auxotrophics (His3, Leu2, Trp1, Ura3)

Pichia Strains ATUM offers Pichia pastoris expression strains for research use, with no licensing restrictions and ready to use with all ATUM Pichia expression vectors.

⬤⬤ PPS-9010 (BG10, wild type): Pichia pastoris wild type expression strain

⬤⬤ PPS-9011 (BG11, aox1Δ (MutS)): A slow methanol utilization derivative, useful for fermentation optimization studies

⬤⬤ PPS-9016 (BG16, pep4Δ, prb1Δ): A protease deficient strain

14 www.atum.bio YEAST EXPRESSION

The effectiveness of a secretion signal depends upon the protein to be secreted. Our panel of vectors with eleven different secretion signals enables you to quickly identify the right leader for your protein. Pichia Secreted Expression

Different proteins express better with different 100 secretion signals LZ 80 SA IN

e AKS AT

da s 60 i FAKS

p t AK

p e KP IV 40 d o n

E AA 20 GA Cutinase and endopeptidase were expressed from Pichia vectors with a methanol inducible AOX1 promoter and different secretion signals. Protein was quantified by 0 0 100 200 300 densitometry of stained acrylamide gels. Cutinase

Saccharomyces Secreted Expression Effectiveness of secretion signal is protein dependent Promoter strength modulates secreted protein expr.

50 80

40 60 FAKS e e SA s s IV a a AKS n d i i 30 t t AK FAKS u p LZ AT

e 40 C AT AK p KP -

o AA

20 H d

D GA IV n AA A E AKS 20 IN 10 LZ SA IN

GA KP 0 0 0 50 100 150 200 0 50 100 150 200 Cutinase TEF-Cutinase Secreted expression of cutinase and endopeptidase from Secreted expression of cutinase from Saccharomyces Saccharomyces vectors with a constitutive TEF promoter vectors with constitutive TEF and ADH promoters and and different secretion signals. Protein was quantified by different secretion signals. Protein was quantified by densitometry of stained acrylamide gels. densitometry of stained acrylamide gels. 15 electra vector system (R)

ATUM has developed a simple, one-tube, universal cloning process that can be performed in a 5-minute bench-top reaction with the fidelity of a restriction based cloning system. Advantages The Electra system uses the type IIS restriction enzyme SapI, which recognizes a 7bp non-palindromic recognition sequence ⬤⬤ Easy and cuts outside of the recognition sequence, leaving a 3bp 1 tube, 5-minute reaction 5’ overhang after digestion. ORFs in a pMOTHER vector can be excised with SapI, resulting in ATG and GGT overhangs. ⬤⬤ Universal Alternatively, PCR can be used to generate ORFs with the any ORF cloned into any Electra vector. correct overhangs. These ORFs can then be easily cloned into Quickly shuttle ORFs from MOTHER pDAUGHTER vectors, which are provided linearized with the vector to multiple DAUGHTER vectors corresponding overhangs.

⬤⬤ Scarless ORF2 Electra site ORF1 Electra site G G T C G A C C T Always in frame with no nucleotide scars A A T ⬤⬤ Choice r p p pDaughter

s

h

L e

pMother / Over 400 vectors with a selection S expression +ORF of fusions, bicistronic expression, vector promoters, RBSs, and more Am p Kan ⬤⬤ Host Systems Bacterial, Mammalian and Yeast amplify / clone ⬤⬤ Convenient Clone your genes using the Electra Electra Reagent Mix Cloning kit, or have ATUM do the work for you mix ⬤⬤ ‘Electra-fy’ your vector 5 min ATUM can modify almost any vector to at room temperature function in the Electra System pMOTHER type IIs sites: >95% transformants with ORF of interest

gene insert

electra mother vector transform

Selection media plates with Kan, +optional Strep or Chloro-phe pDAUGHTER overhangs (linearized vector): Single tube digestion and ligation reaction using the Electra Vector System. The pMOTHER vector, carrying the gene of gene insert interest, is mixed with linearized pDAUGHTER vector in the presence of Electra Reagent Mix. The reaction is incubated for 5 minutes at room temperature, transformed into E. coli electra daughter vector and plated on LB/agar plates with antibiotic. Simple, scarless cloning.

16 www.atum.bio ELECTRA SYSTEM & GENE GPS

geneGPS expression optimization (R)

Not all codon optimization algorithms are created equal. Genes optimized with GeneGPS algorithms for maximal expression yield between 10 and 100 fold more protein. Save months of time on painstaking optimization studies and use much smaller culture volumes.

1. in silico DoE 2. gene synthesis 3. test in host 4. build predictive infologs system model Interrogating host preferences using multivariate regression expression over both wt and traditional codon optimization (e.g., PLS) and sequence space exploration is used to develop methods. Multiple rounds provide magnitudes of functional GeneGPS algorithms, providing significantly increased improvement. PLoS ONE 2009, 4(9):e7002, Welch et al.

Host Systems GeneGPS algorithms can optimize expression in your host. ATUM is using the results of our research to create patented gene design algorithms for numerous host systems. Different symbols/colors represent different proteins.

Baculovirus/Sf9 S. cerevisiae Pichia pastoris E. coli Measured Expression Measured

Predicted Expression Mammalian Clostridium Plant Fungus (CHO and HEK293) Measured Expression Measured

Predicted Expression 17 expression services

Every gene and protein construct is different. Every Large Scale Plasmid Preps research lab has unique protocols and methods. Receive your synthetic genes in sufficient E. coli How do you know if your gene expression in quantities for immediate experimentation. ATUM is optimal? Large Scale Plasmid Prep service brings you convenience, time savings, and high quality DNA. Expression Test in E. coli Simply tell us the yield your research requires and Quickly know what expression levels to expect we will determine optimal volumes and conditions with your synthetic gene. No more questioning if to provide the DNA you need. your gene was unable to express or if difficulties The DNA is of high homogeneity and is available were due to protocols followed. ATUM will clone at low or very low endotoxin grades. The quality and test your gene for expression levels in E. coli. certificate includes double strand DNA sequencing and DNA yield determination. Benefits ⬤⬤ Available for bacterial vectors & vector panels Yield Grade* ⬤⬤ ATUM Expression Data Report 10 - 100 µg Low endotoxin ⬤⬤ SDS-PAGE and/or semi-quantitative Western 100 µg - 500 µg Very low endotoxin results 500 µg - 2 mg Very low endotoxin ⬤⬤ Growth and induction protocols 2 - 10 mg Very low endotoxin

Gene Synthesis * Low endotoxin levels are sufficient for popular production cell lines (HEK, CHO, etc.). Very low endotoxin levels (gener- ⬤⬤ Flexibility of design to add tags, restriction ally < 0.1 EU/µg DNA) are ideal for primary cell culture. Endo- sites, promoters, and more toxin levels in all plasmid preps are not measured by ATUM. ⬤⬤ Patented GeneGPS expression optimization

⬤⬤ Cloning into any vector

⬤⬤ Gene variants and libraries

⬤⬤ Ph.D. level customer support From idea to bench Custom Cloning in as little as 5 days. Your gene in your vector. Simply provide the custom vector and we’ll handle the cloning and sequencing. ATUM fully protects your IP and confidentiality at all times; and of course, custom vectors remain your property throughout the cloning process. Your vector will always be available to your account for any future orders.

18 www.atum.bio EXPRESSION SERVICES & VECTOR GPS vectorGPS(R)

ITR polyA Create the vector that works best in your beta globin expression system. VectorGPS uses Design-of- BGH HGH Experiment (DoE) algorithms to build testable Backbone numbers of vectors from sets of control elements, RNA Export HPRE and advanced machine learning to assess AGS WPRE the contribution of each element to vector SAR performance. Critical vector elements perform differently in different systems, so instead of using vectors packed with fossil sequences put there by graduate students twenty years ago for reasons VectorGPS no one remembers, let ATUM help you create the custom vector you need. ORF Systematic sampling of mammalian vector elements. SV40 ori Intron EBV oriP CMVc Machine learning algorithms identified vector SV40 large T-ag EF1a Promoter eMLP EBNA Enhancer EF1a components that contributed to expression of EF1a actin CMV ITR CMV DasherGFP in HEK293 (red bars) or CHO (blue GAPDH bars) cells. Positive contributions have positive Actin Mammalian vector systems in particular, have a large regression weights, the magnitude of the weight number of vector elements that contribute to vector corresponds to the magnitude of the effect of the performance. Viral vectors such as lentiviral and adeno- element. associated viral (AAV) vectors can suffer from additional constraints of sequence and size. We have identified vector elements in bacterial, mammalian, lentiviral and adeno- associated viral systems that show enhanced expression and/or integration properties.

5´ UTR elements replication promoters, enhancers, introns 3´ UTR elements elements

0.8 0.6 0.4 0.2 0 -0.2 -0.4

Model Regression Weight -0.6 -0.8 CHO HEK293

19 research. create. break through.

+1 877 DNA TOGO +1 650 853 8347 [email protected]