Editing S30 E. coli extract (section)

== Protocol 6 ==



https://www.researchgate.net/publication/259270251_Synthesis_of_23_mgmL_of_Protein_with_an_all_E_coli_Cell-Free_Transcription-Translation_System

The crude extract was prepared as above<br/>using the BL21 Rosetta2 strain and with the following slight modifications.<br/>S30A buffer composition: 14 mM Mg-glutamate, 60 mM<br/>K-glutamate, 50 mM Tris buffered with acetic acid to pH 8.2. S30B<br/>buffer composition: 14 mM Mg-glutamate, 150 mM K-glutamate<br/>buffered to pH 8.2 with Tris. The lysate was prepared with a cell<br/>press at a pressure of 16,000 LB.<br/>2.2. Cell-free reaction in batch mode<br/>The reaction mixture was prepared as described before [20].<br/>The reaction buffer is composed of: 50 mM Hepes pH 8, 1.5 mM<br/>ATP and GTP, 0.9 mM CTP and UTP, 0.2 mg/ml tRNA, 0.26 mM<br/>coenzyme A, 0.33 mM NAD, 0.75 mM cAMP, 0.068 mM folinic acid,<br/>1 mM spermidine, 30 mM 3-PGA, 1 mM DTT, 2% PEG8000. A<br/>typical cell-free reaction is composed of 33% (volume) of E. coli<br/>crude extract. The other 66% of the reaction volume are composed<br/>of the plasmids and the reaction buffer containing the nutrients.<br/>The amino acid concentration was adjusted to either 1.5 mM or<br/>3 mM of each of the 20 amino acids. Mg-glutamate and K-glutamate<br/>concentrations were adjusted according to the plasmids used<br/>(60 mM K-glutamate and 5 mM Mg-glutamate for pBEST-OR2-<br/>OR1-Pr-UTR1-deGFP-T500, 20 mM K-glutamate and 5 mM Mgglutamate<br/>for the T7 cascade). The concentration of synthesized<br/>deGFP was determined from a calibration curve (Ex 485 nm and<br/>Em 528 nm) done with pure recombinant eGFP (reGFP) purchased<br/>from Cell Biolabs Inc. End-point fluorescence was measured in a<br/>384 format well plate using a BioTek plate reader. deGFP synthesis<br/>kinetics were measured in a 96-well plate sealed with a plastic cap<br/>to avoid evaporation.





https://www.future-science.com/doi/full/10.2144/000114249

The 20 solid amino acids are dissolved at concentrations between 1.6 and 4 M in 5 M KOH, and mixtures containing each<br/>of the 20 amino acids are prepared for cell-free expression. The mixtures are diluted in water to achieve a total amino acid<br/>concentration between 253 and 268 mM. The pH of the resulting aqueous solutions is adjusted with acetic acid. The amino<br/>acid mixtures are frozen in liquid nitrogen and stored at -80°C.&nbsp;<span style="font-family: Roboto, sans-serif; font-size: 15px;">The 20 solid amino acids were dissolved in 5 M KOH to produce concentrations of 1.6–4 M. These stock solutions, stored at −20°C in a liquid phase, can be used for a few weeks.&nbsp;&nbsp;mixture stability depended on amino acid concentration, composition, and pH.&nbsp;On average, amino acid mixtures prepared with this method were 30 times less expensive than those prepared with an amino acid sampler kit.&nbsp;The mixtures were stable at room temperature (RT; ∼25°C), during aliquoting (∼20 min), and when snap frozen in liquid nitrogen for long storage at −80°C (∼6 months).&nbsp;</span><span style="font-family: Roboto, sans-serif; font-size: 14px;">deGFP was synthesized through the T7 cascade with 0.1 nM pBEST-OR2-OR1-Pr-UTR1-T7-T500 plasmid, 1 nM pIVEX2.3d-deGFP plasmid, 20 mM K-glutamate, and 2 mM Mg-glutamate.&nbsp;</span><span style="font-family: Roboto, sans-serif; font-size: 14px;">The synthesis was carried out using three different amino acid distributions: (A) constant, (B)&nbsp;</span>''E. coli''<span style="font-family: Roboto, sans-serif; font-size: 14px;">, and (C) deGFP sequence-based (1 mg/mL deGFP = 39.39 µM).&nbsp;&nbsp;</span>For the constant distribution (Figure 1A), the highest yield reached was 83 µM, which is equal to 2.11 mg/mL of active protein. This was obtained with a mixture containing ∼2.75 mM of each of the amino acids at pH 6.51. For the E. coli distribution, leucine is the most abundant amino acid (10.5%) and cysteine the least abundant (1.1%). The maximum deGFP synthesis was 1.55 mg/mL (∼61 µM) with amino acids stabilized at pH 6.04. The last concentration range was measured for the deGFP distribution mixture. Glycine (8.9%) and leucine (8.4%) were the most abundant, whereas cysteine (0.9%) and tryptophan (0.4%) were the least abundant. A protein yield of 1.35 mg/mL (∼53 µM) was measured at pH 6.82.&nbsp; L<span style="font-family: Roboto, sans-serif; font-size: 15px;">arger protein yields were reached at slightly acidic pHs between 6.0 and 6.8 and&nbsp;the highest production of active deGFP was observed with the constant distribution mixture</span><span style="font-family: Roboto, sans-serif; font-size: 14px;"></span><span style="font-family: Roboto, sans-serif; font-size: 15px;"></span>

The reporter protein deGFP, a slightly modified version of eGFP (10), was expressed through a transcriptional activation cascade using the following two plasmids (1): pBEST-p15A-OR2-OR1-Pr-T7rnap-T500 (0.1 nM) to express the T7 RNA polymerase and pIVEX2.3d-deGFP (1 nM)
<br/>
Briefly, BL21 Rosetta2 cells are washed with S30A buffer: 14 mM Mg-glutamate, 60 mM K-glutamate, 50 mM Tris, and 2 mM DTT, buffered with glacial acetic acid to pH 8.2. The cytoplasm is extracted using a cell-press (12000 LB) and clarified by centrifugation at 30,000 × g. Then the cellular extract is dialyzed at 4°C for 1 h against the S30B buffer (14 mM Mg-glutamate and 150 mM K-glutamate, buffered to pH 8.2 with 2 M Tris) using cassettes with a 10 kDa cut-off. The crude extract represents 33% (30 µL) of the final reaction volume (90 µL), giving roughly 9.2 mg/mL of protein, and the remaining volume has the following components: water, 2 mM Mg-glutamate, 20 mM K-glutamate, 2% PEG-8000 (v/v), amino acids, maltodextrin at 216.186 g/L, and the reaction buffer (RB). The RB in the cell-free reactions has the following components: 50 mM Hepes pH 8, 1.5 mM ATP and GTP, 0.9 mM CTP and UTP, 0.2 mg/mL tRNA, 30 mM 3-PGA, 0.26 mM CoA, 0.33 mM NAD, 0.75 mM cAMP, 0.068 mM folinic acid, and 1 mM spermidine

L-Amino acids

[https://www.sigmaaldrich.com/catalog/product/sial/laa21?lang=en&region=US https://www.sigmaaldrich.com/catalog/product/sial/laa21?lang=en&region=US], $<span style="color: rgb(77, 77, 77); font-family: Arial, Helvetica, sans-serif; font-size: 12px; text-align: right;">383</span>

4 amino acids: (trans-4-hydroxy-l-proline, L-cysteine hydrochloride, L-histidine hydrochloride, L-lysine hydrochloride) should be replaced by:&nbsp;L-proline Sigma 81709–25G,&nbsp; L-cysteine Sigma 30089–25G,&nbsp; L-histidine Sigma 53319–25G, L-lysine Sigma L5501–5G

&nbsp;&nbsp;Table S1 Amino acid preparation at equimolar concentrations.&nbsp;



{|
|-
| width="64" | 
| width="64" | MW
| width="64" | mM
| width="64" | µL AA mix
| width="64" | mM in KOH mixture
|-
| Alanine
| 89.09
| 4089
| 13.6
| 146
|-
| Arginine*
| 210.67
| 2314
| 22.2
| 135
|-
| Asparagine
| 132.12
| 3759
| 13.6
| 134
|-
| Asparticacid
| 133.1
| 3752
| 13.6
| 134
|-
| Cysteine
| 121.16
| 2465
| 22.2
| 143
|-
| Glutamicacid
| 147.13
| 3655
| 13.6
| 130
|-
| Glutamine
| 146.15
| 2501
| 22.2
| 145
|-
| Glycine
| 75.07
| 4210
| 13.6
| 150
|-
| Histidine
| 155.15
| 3281
| 13.6
| 117
|-
| Isoleucine
| 131.18
| 3765
| 13.6
| 134
|-
| Leucine
| 131.18
| 2549
| 22.2
| 148
|-
| Lysine
| 146.19
| 2392
| 22.2
| 139
|-
| Methionine
| 149.21
| 2491
| 22.2
| 145
|-
| Phenylalanine
| 165.19
| 1716
| 34
| 153
|-
| Proline
| 115.13
| 3883
| 13.6
| 138
|-
| Serine
| 105.9
| 3953
| 13.6
| 141
|-
| Threonine
| 119.12
| 3853
| 13.6
| 137
|-
| Tryptophan
| 204.23
| 1661
| 34
| 148
|-
| Tyrosine
| 181.19
| 2396
| 22.2
| 139
|}

All of the solids (LAA21–1KT) are purchased from Sigma-Aldrich (St. Louis, MO) and dissolved according&nbsp;to the concentration listed in the table above in 500 µL 5 M KOH solution

Note that the final concentration of the amino acid stocks solution is calculated to account for&nbsp;a volume increase due to the physical mass of the solid. As an estimation, the volume increases is half&nbsp;the mass of the solid.<br/>*Arginine monohydrocloride is dissolved in water. All of the other L-amino acids that have been used do&nbsp;not have counter ions.<br/>To 381.6 µL of equimolar amino acids mixture at 2807 mM in 5 M KOH, water is added up to ~4 mL,&nbsp;and the pH is adjusted with glacial acetic acid:<br/>• 100 µL acetic acid, pH = 8.49; the mixture is stable during aliquoting.<br/>• 105 µL acetic acid, pH = 7.86; the mixture is stable during aliquoting.<br/>• 110 µL acetic acid, pH = 6.51; the mixture is stable during aliquoting.

Table S2. Amino acids preparation according to the E. coli distribution (4)



{| cellspacing="0" cellpadding="0"
|-
| AA
| E. coli distribution&nbsp;%<br/>
| mM in KOH mixture<br/>
| µL AA mix<br/>
|-
| Alanine
| 9.7
| 280
| 26
|-
| height="19" | Arginine
| 5.3
| 154
| 25
|-
| height="19" | Asparagine
| 3.6
| 104
| 11
|-
| height="19" | Aspartic acid
| 4.8
| 139
| 14
|-
| height="19" | Cysteine
| 1.1
| 31
| 5
|-
| height="19" | Glutamic acid
| 5.4
| 156
| 16
|-
| height="19" | Glutamine
| 6.2
| 177
| 27
|-
| height="19" | Glycine
| 7.7
| 222
| 20
|-
| height="19" | Histidine
| 1.9
| 54
| 6
|-
| height="19" | Isoleucine
| 5.5
| 158
| 16
|-
| height="19" | Leucine
| 10.8
| 311
| 47
|-
| height="19" | Lysine
| 4.3
| 125
| 20
|-
| height="19" | Methionine
| 2.9
| 84
| 13
|-
| height="19" | Phenylalanine
| 4.1
| 119
| 27
|-
| height="19" | Proline
| 4.2
| 122
| 12
|-
| height="19" | Serine
| 5.5
| 158
| 15
|-
| height="19" | Threonine
| 5.1
| 146
| 14
|-
| height="19" | Tryptophan
| 1.5
| 44
| 10
|-
| height="19" | Tyrosine
| 2.7
| 78
| 12
|-
| height="19" | Valine
| 7.5
| 215
| 32
|}



All of the solids (LAA21–1KT) are purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved according to the concentrations listed in the table below in 500 µL 5 M KOH solution purchased from Sigma-Aldrich. Amino acids are dissolved in 5 M KOH as described in Supplementary Table S1.

*Arginine monohydrocloride is dissolved in water. All the others L-amino acids that have been used do not have counter ions.

To 369 µL of E. coli amino acids mixture at 2878 mM in 5 M K(OH), water is added up to ~4 mL, and the pH adjusted with glacial acetic acid:

• 95 µL acetic acid, pH = 8.21; the mixture is stable during aliquoting.

• 100 µL acetic acid, pH = 7.51; the mixture is stable during aliquoting.

• 110 µL acetic acid, pH = 6.04; the mixture is stable during aliquoting.



Table S3. Amino acids mixture preparation according to deGFP sequence.&nbsp;

{| border="0" cellspacing="0" cellpadding="0"
|-
| AA
| deGFP distribution&nbsp;%<br/>
| mM in K(OH) mixture&nbsp;<br/>
| &nbsp;µL AA mix<br/>
|-
| Alanine
| 3.7
| 105
| 10
|-
| height="19" | Arginine
| 2.6
| 73
| 12
|-
| height="19" | Asparagine
| 5.5
| 156
| 16
|-
| height="19" | Aspartic acid
| 7.2
| 203
| 21
|-
| height="19" | Cysteine
| 0.9
| 26
| 4
|-
| height="19" | Glutamic acid
| 5.7
| 162
| 17
|-
| height="19" | Glutamine
| 3.7
| 105
| 16
|-
| height="19" | Glycine
| 9.5
| 267
| 24
|-
| height="19" | Histidine
| 3.3
| 94
| 11
|-
| height="19" | Isoleucine
| 5.1
| 142
| 14
|-
| height="19" | Leucine
| 8.9
| 249
| 37
|-
| height="19" | Lysine
| 7.9
| 223
| 36
|-
| height="19" | Methionine
| 2.3
| 64
| 10
|-
| height="19" | Phenylalanine
| 5.8
| 162
| 36
|-
| height="19" | Proline
| 4.3
| 122
| 12
|-
| height="19" | Serine
| 4
| 113
| 11
|-
| height="19" | Threonine
| 6.5
| 184
| 18
|-
| height="19" | Tryptophan
| 0.4
| 12
| 3
|-
| height="19" | Tyrosine
| 4.4
| 123
| 20
|-
| height="19" | Valine
| 8.2
| 230
| 34
|}

The composition is retrieved from the gene sequence using [http://www.expasy.org/ http://www.expasy.org/]. All of the solids (LAA21–1KT) are purchased from Sigma-Aldrich (St. Louis, MO) and dissolved according to the concentrations listed in the table below in 500 µL 5 M KOH solution purchased from Sigma-Aldrich. Amino acids are dissolved in 5 M KOH as described in Supplementary Table S1.

*Arginine monohydrocloride is dissolved in water. All of the other L-amino acids that have been used do not have counter ions.

To 360 µL of the E. coli amino acids mixture at 2812 mM in 5 M K(OH), water is added up to ~4 mL, and the pH is adjusted with glacial acetic acid:

• 95 µL acetic acid, pH = 8.72; the mixture is stable during aliquoting.

• 100 µL acetic acid, pH = 7.9; the mixture is stable during aliquoting.

• 105 µL acetic acid, pH = 6.82; the mixture is stable during aliquoting.