Bacterial expression vector pGEX-4T1 (GE Healthcare, Munich, Germany) was used as a template to generate mutated and truncated human FAK as GST (Glutathione S-transferase) fusion proteins. Point mutations (L113A, P116C, P116G, P116N, P117K, and P116S) and triple mutants (L113A/P116N/P117K, L113A/P116C/P117G, L113A/P116A/P117A) were generated using the Quick Change II XL Site-Directed Mutagenesis kit (Agilent Technologies, (Santa Clara, CA). Truncations were generated through PCR using forward and reverse primers to direct truncation (Table 1 ). PCR products were introduced into the pGEX-4T1 template between 5′ EcoRI and 3′XhoI sites. Plasmids were purified via MiniPrep (QIAGEN, Valencia, CA) before sequencing. BL21 competent E. coli (New England Biolabs, Ipswich, MA) were transformed with appropriate plasmids, and IPTG-induced.
Bl21 competent e coli
Manufactured by New England Biolabs
BL21 competent E. coli is a laboratory strain of Escherichia coli bacteria that is commonly used for the expression of recombinant proteins. It is designed to support high-level protein expression from T7-based expression systems.
Automatically generated - may contain errors
4 protocols using bl21 competent e coli
1
Generation of Mutant and Truncated FAK Constructs
2
Recombinant UGT76G1 Enzyme Production
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Example 9
Preparation and Activity of UGT76G1 Prepared by pMAL Plasmid and BL21 Expression Strain
After subcloning the synthetic UGT76G1 gene into the pMAL plasmid using NdeI and SalI cloning sites, the pMAL_UGT76G1 plasmid was transformed into BL21 expression strain (New England Biolabs BL21 Competent E. coli) by heat shock treatment. The obtained cells were grown on LB Agar medium in petri-dishes in the presence of Ampicillin. Suitable colonies were selected and allowed to grow in liquid LBGKP medium containing Ampicillin). Glycerol was added and 400 μL aliquots were stored at −20° C. and at −80° C.
A storage aliquot was thawed and added to 30 mL of LBGKP medium. This culture was allowed to shake at 30° C. for 8 h. and subsequently used to inoculate 400 mL of production medium containing 60 g/L of “Overnight express instant TB medium” (Novagen, reference 71491-5), 10 g/L of glycerol and 50 mg/L of Ampicillin. The medium was allowed to stir at 20° C. while taking samples to measure the OD and pH. After 40 h, the cells were harvested by centrifugation and frozen. The obtained cell wet weight was 5.86 g.
2.74 g of obtained pellet was lysed by addition of 9.6 mL of “Bugbuster Master Mix” (Novagen, reference 71456) and 4.1 mL of water. The lysate was recovered by centrifugation and kept frozen.
3
Recombinant B. subtilis Spores Expressing NK-lysin
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Recombinant B. subtilis spores expressing empty vector (B. subtilis-EV) or B. subtilis-cNK-2 were constructed and provided by US Biologic (Memphis, TN). The NK-lysin used for the expression in a bacterial vector was based on the chicken NK-lysin sequence (RRQRSICKQLLKKLRQQLSDALQNNDD) reported previously (1 (link), 9 (link)), which was then cloned into the pTTB2 expression vector (MoBiTec). Briefly, pTTB2-cNK was then expanded in BL21 competent E. coli (New England Biolabs, Inc., Ipswich, MA) and purified using the GeneJET Plasmid Miniprep Kit (Thermo Fischer Scientific, Madison, WI). Sequences were confirmed using Sanger sequencing and purified plasmids were religated using the Rapid DNA Ligation (Thermo Fisher Scientific, Madison, WI). Competent B. subtilis cells (strain WB800N, MoBiTec) were transformed using 0.1 M EGTA and expanded on agar plates using 2% xylose as a selection agent. Single colonies were sequenced and expanded using 2xYT media (Difco, BD Diagnostic Systems, Sparks, MD).
4
Recombinant UGT76G1 Protein Production
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Example 9
Preparation and Activity of UGT76G1 Prepared by pMAL Plasmid and BL21 Expression Strain
After subcloning the synthetic UGT76G1 gene into the pMAL plasmid using NdeI and Sal1 cloning sites, the pMAL_UGT76G1 plasmid was transformed into BL21 expression strain (New England Biolabs BL21 Competent E. coli) by heat shock treatment. The obtained cells were grown on LB Agar medium in petri-dishes in the presence of Ampicillin. Suitable colonies were selected and allowed to grow in liquid LBGKP medium containing Ampicillin). Glycerol was added and 400 μL aliquots were stored at −20° C. and at −80° C.
A storage aliquot was thawed and added to 30 mL of LBGKP medium. This culture was allowed to shake at 30° C. for 8 h. and subsequently used to inoculate 400 mL of production medium containing 60 g/L of “Overnight express instant TB medium” (Novagen, reference 71491-5), 10 g/L of glycerol and 50 mg/L of Ampicillin. The medium was allowed to stir at 20° C. while taking samples to measure the OD and pH. After 40 h, the cells were harvested by centrifugation and frozen. The obtained cell wet weight was 5.86 g.
2.74 g of obtained pellet was lysed by addition of 9.6 mL of “Bugbuster Master Mix” (Novagen, reference 71456) and 4.1 mL of water. The lysate was recovered by centrifugation and kept frozen.
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