TITLE: Cell-free extract,  4x Wizard mix and CFPS reaction preparation- Haseloff Lab
AUTHORS: Fernando Guzman Chavez, Jim Haseloff

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Following this recipe, you will cell-free extracts to perform Tx-TL reactions using plasmid or linear DNA based on P70a and T7 promoters. Likewise, this protocol describes how to prepare 4x wizard mix and setting up a CFPS reaction.</div><div class = "text-block">NOTE :</div><div class = "text-block">The protocol described here is an adaptation from these papers:</div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;"><span style = "font-weight:bold;">Adam D. Silverman</span><span>, Nancy Kelley-Loughnane, Julius B. Lucks, and Michael C. Jewett (2019).</span><span style = "font-style:italic;">Deconstructing Cell-Free Extract Preparation for in Vitro Activation of Transcriptional Genetic Circuitry</span><span>. ACS Synthetic Biology, 403-414. DOI: 10.1021/acssynbio.8b00430.</span></li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><span style = "font-weight:bold;">Andriy Didovyk</span><span>, Taishi Tonooka, Lev Tsimring, and Jeff Hasty. (2017).</span><span style = "font-style:italic;">Rapid and Scalable Preparation of Bacterial Lysates for Cell-Free Gene Expression</span><span>. ACS Synthetic Biology, 2198-2208. DOI: 10.1021/acssynbio.7b00253.</span></li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><span style = "font-weight:bold;">Yang WC,</span><span> Patel KG, Wong HE, Swartz JR.(2012). </span><span style = "font-style:italic;">Simplifying and streamlining Escherichia coli-based cell-free protein synthesis</span><span>.Biotechnol Prog. 28(2):413-420. DOI:10.1002/btpr.1509.</span></li></ul></div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;"><span style = "font-weight:bold;">Andriy Didovyk</span><span>, Taishi Tonooka, Lev Tsimring, and Jeff Hasty. (2017).</span><span style = "font-style:italic;">Rapid and Scalable Preparation of Bacterial Lysates for Cell-Free Gene Expression</span><span>. ACS Synthetic Biology, 2198-2208. DOI: 10.1021/acssynbio.7b00253.</span></li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><span style = "font-weight:bold;">Yang WC,</span><span> Patel KG, Wong HE, Swartz JR.(2012). </span><span style = "font-style:italic;">Simplifying and streamlining Escherichia coli-based cell-free protein synthesis</span><span>.Biotechnol Prog. 28(2):413-420. DOI:10.1002/btpr.1509.</span></li></ul></div><div class = "text-block"><ul style = "list-style-type:disc;"></ul></div><div class = "text-block">Please note that some steps were taken literally as they appear in these sources, and it is highly recommended to read  these papers before starting. </div><div class = "text-block">Our adaptations includes: Changes in buffers, volumes to perform the cell-free reactions using 12 µL as final volume.</div></div>

[STEPS]
?. [Preparing cell extracts]
5 mL started culture (BL21 DE Star) were grown overnight at 37°C from a single colony in LB plate.
?. [Preparing cell extracts]
At the next day 50 mL of LB medium were inoculated with 500 µL of the stationary culture from step 1 and grown at overnight at 37°C.
?. [Preparing cell extracts]
At day 3, 12 mL of the stationary culture were inoculated into 400 mL of 2xYTG and grown at 37°C on an orbital shaker at 200 rpm in a 2.5 L baffled Tunair flask.
?. [Preparing cell extracts]
Culture were grown to the exponential phase optical density (OD600) 2.5-3.0 for approximately 4h. # If you are planning to use plasmid/ linear DNA based on T7 promoter, the culture needs to be induced with 400µL 1 M IPTG when it reaches an OD of 0.6.
?. [Preparing cell extracts]
Quickly, chill the cells on water/ice bath and incubate on ice for 15 min. #From now on everything has to be performed on ice.
?. [Preparing cell extracts]
Split the culture in two and centrifugate for 12 min at 5000g at 4°C to pellet the cell. # Use 250 mL centrifuge bottles.
?. [Preparing cell extracts]
Wash the 3 times the pellets with each time resuspending them in the buffer: two times with cold TBST and the last with cold S30 buffer supplemented with 2 mM dithiothreitol (DTT). Every time for 12 min at 5000g at 4°C.
?. [Preparing cell extracts]
Keep and flash frozen the pellet in a pre-weighted 50mL falcon tube. # The pellet can be stored at -80°C from 1 to 2 weeks.
?. [Preparing cell extracts]
Before opening the cells, weight the pellet and resuspend into cold S30 buffer supplemented with 2 mM DTT. #You have to know the weight as you do not want to overdilute the lysate. Rule of thumb is going for 1g of cells/0.9ml of buffer.
?. [Preparing cell extracts]
Open cell quickly with Emulsiflex ~17500-25000 psi and collect the lysate, overall suspension must be passed once. # Wait until pressures changes are stabilized. Alternatively, you can use another cell disruption system as sonication.
?. [Preparing cell extracts]
Alternatively, to lysate the cell suspension by sonication, use a QSonica Q125 sonicator with a 3.175 mm diameter probe at a frequency of 20 kHz and 50% amplitude by 10 s ON/OFF pulses for a total of 60 s (delivering∼350 J).
?. [Preparing cell extracts]
Transfer the lysate into chilled tubes and centrifuge for 30min at 30 000 g and 4°C, collect 2/3 of the supernatant and repeat the procedure.
?. [Preparing cell extracts]
Then, cell lysate was aliquoted, flash frozen and stored at -80°C.
?. [Preparing cell extracts]
A) Alternatively, a runoff reaction can be performed by transferring the lysate into a falcon tube and incubate at 37°C for 80 min at 200 rpm in dark conditions. Following this incubation, the extract was centrifugated for 10 min at 4°C and 12000g. Then, supernatant was aliquoted, flash frozen and stored at -80°C.
?. [Preparing cell extracts]
B) Alternatively, a runoff reaction can be performed by transferring the lysate into a falcon tube and incubate at 37°C for 80 min at 200 rpm in dark conditions. Following this incubation, the extract was centrifugated for 10 min at 4°C and 12000g. Then, supernatant was removed and injected into 10K MWCO and dialyzed against 600 mL of S30 Dialysis buffer. After, the extract was centrifugated for 10 min at 4°C and 12000g. Supernatant was aliquoted, flash frozen and stored at -80°C.
?. [Preparing 4X Wizard mix]
To prepare 500 µL of 4X Wizard mix, thaw the aliquots of each precursor solution and pipette the required volume following the order in the next table.  AB1Precursor solutionRequired quantity (µL)21)	Autoclaved MQ water4032)	1000mM Magnesium glutamate20             # for a final concentration of 10mM in CFPS43)	10x Salt Solution mix20054)	25x Nucleotide mix8065)	25x 19 Amino Acid Mix8076)	25x PEP80
AB1Precursor solutionRequired quantity (µL)21)	Autoclaved MQ water4032)	1000mM Magnesium glutamate20             # for a final concentration of 10mM in CFPS43)	10x Salt Solution mix20054)	25x Nucleotide mix8065)	25x 19 Amino Acid Mix8076)	25x PEP80
?. [Performing cell-free reactions]
To set up a single cell-free reaction of 12µL, thaw the aliquots of each precursor solution and pipette the required volume following the order in the next table.# Cell-free extract must be added always at the end.  AB1Precursor solutionRequired quantity (µL)21)	Autoclaved MQ water1.432)	40% PEG 80000.643)	DNA (20 nM)354)	4x Wizard mix365)	Cell extract47Final Volume12
AB1Precursor solutionRequired quantity (µL)21)	Autoclaved MQ water1.432)	40% PEG 80000.643)	DNA (20 nM)354)	4x Wizard mix365)	Cell extract47Final Volume12
?. [Commentaries]
To prepare the all solutions described in this file, see the adequate version from protocol " Solutions for CFPS".
?. [Literature/ References]
?. [Change history]
V1-0 →V1-1 Protocol to cell disruption by sonication was added.V1-1→ DNA concentration in protocol " Performing cell-free reactions" was corrected