Transformation


The transformation efficiency was calculated using the following equation:


Competent cells prep protocol optimization

-Different buffers were used to prepare competent cells to test their effect on the transformation efficiency using DH5-alpha cells and optimize the best protocol for our experiments.
-Effect of OD600 on the transformation efficiency.
The tested buffers were; TSS, CaCl2 & MgCl2/CaCl2.


TSS Buffer
CaCl2 Buffer
CaCl2/ MgCl2 Buffer
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Description


The buffer requires a small number of chemical components that may be added as one reagent rather than several additions and does not require several washing or incubation procedures, it does not require a heat shock step, and the prepared cells can be frozen directly and stored for future use which makes this procedure convenient.


Protocol overview


Bacteria are grown to the early exponential phase and then treated with a transformation and storage solution (TSS) comprising polyethylene glycol (PEG), dimethyl sulfoxide (DMSO), and magnesium. The approach is a one-step procedure. Competent cells are then combined with plasmid DNA, held on ice for 5–30 min, then incubated at 37 ° to facilitate the production of an antibiotic resistance gene, after which the mixture is plate-incubated.

Protocol link

Results of transformation



- OD600 before comptency= 0.35
- No. of colonies = 2370
- Vol. of culture plated = 25µL
- Transformation efficiency = 9.48 x 10^5 transformants /µg
- CFU/mL = 1.896 x 10^6




- OD600 before competency= 0.45
- No. of colonies = 3660
- Vol. of culture plated = 25µL
- Transformation efficiency = 1.344 x 10^6 transformants per µg
- CFU/mL = 2.928 x 10^6

Comment


results shows a higher transformation efficiency at OD600 = 0.45 using TSS buffer.

Description


CaCl2 is the most frequently used buffer to prepare competent cells.
Plasmid DNA can attach to lipopolysaccharide (LPS) receptor molecules when calcium chloride is added to the cell suspension. Thus, when heat shock is applied, the negatively charged DNA backbone and LPS combine, and plasmid DNA enters the bacterial cell.


Protocol overview


in the CaCl2 method, competency can be obtained by creating cracks in bacterial cells by suspending them in a solution containing a high concentration of calcium. DNA can then be forced into the Host cell by heat shock treatment at 42°C for the process of transformation.

Protocol link

Results of transformation



- OD600 before competency= 0.6
- No. of colonies = 277 colony
- Vol. of culture plated = 25µL
- Transformation efficiency = 1.108 x 10^5 transformants /µg
- CFU/mL = 2.216 x 10^5



- OD600 before competency= 0.6
- No. of colonies = 700 colony
- Vol. of culture plated = 100µL
- Transformation efficiency = 7 x 10^4 transformants /µg
- CFU/mL = 1.4 x 10^5



- OD600 before competency= 1.6
- No. of colonies =505 colonies
- Vol. of culture plated=25µL
- Transformation efficiency = 2.02 x 105 transformants per µg
- CFU/mL = 4.04 x 10^4



- OD600 before competency= 1.6
- No. of colonies = 160 colony
- Vol. of culture plated = 100µL
- Transformation efficiency = 1.6 x 10^4 transformants per µg
- CFU/mL = 3.2 x 10^4


Comment


The results shows a higher transformation efficiency at OD600 = 1.6 using CaCl2 buffer.

Description


MgCl2 behaves similarly to CaCl2. Changing the permeability of the membranes, it causes the cells to uptake DNA.

Protocol overview

just as the CaCl2 method, the competency is achieved chemically by the use of a solution containing a high concentration from both cations; ca2+ & Mg2+ in the form of CaCl2 & MgCl2 and then DNA can be forced into the Host cell by a heat shock treatment at 42°C for the process of transformation.

Protocol link

Results of transformation



-OD600 before competency= 0.4
- No. of colonies = 110
- Vol. of culture plated = 25ul
- Transformation efficiency = 4.4 x 10^4 transformants /µg
- CFU/mL = 8.8 x 10^4


- OD600 before competency= 0.4
- No. of colonies = 180
- Vol. of culture plated = 100ul
- Transformation efficiency = 1.8 x 10^4 transformants /µg
- CFU/mL = 3.6 x 10^4


- OD600 before competency= 0.9
- No. of colonies = 270
- Vol. of culture plated = 25ul
- Transformation efficiency = 1.08 x 10^5 transformants /µg
. - CFU/mL = 2.16 x 10^5


- OD600 before competency= 0.9
- No. of colonies = 500
- Vol. of culture plated = 100ul
- Transformation efficiency = 5 x 10^4 transformants /µg
- CFU/mL = 1 x 10^5

Comment

The results show a higher transformation efficiency at OD600 = 0.9 using CaCl2 buffer


Comment

The results show a transformation efficiency of 1.08 x 10^5 transformants per µg for CaCl2/MgCl2 protocol, 2.02 x 10^5 transformants per µg for CaCl2 protocol and 1.344 x 10^6 transformants per µg using TSS protocol.

Accordingly, TSS buffer was considered to be the best choice to work with followed by CaCl2.

The effect of bacterial recovery step on the transformation efficiency using TSS & CaCl2 buffers

TSS Buffer Vs CaCl 2 buffer

Description


Competent cells prepared using TSS and CaCl2 buffers showed the highest transformation efficiency, we further tested their ability to maintain high transformation efficiencies without the need for a recovery step, using DH5-alpha.


TSS buffer



- No. of colonies = 173 colony
- Vol. of culture plated = 25µL
- Transformation efficiency = 1.3840 x 10^4 transformants per µg
- CFU/mL = 1.3840 x 10^4

CaCL2



- No. of colonies = 8 colonies
- Vol. of culture plated = 10µL
- Transformation efficiency = 1600 transformants per µg
-CFU/mL = 1600


Comment


The result shows a higher ability of the cells transformed with TSS to skip the recovery step

TSS buffer protocol optimization

The high transformation efficiencies of TSS buffer encouraged us to optimize the protocol conditions to get even higher transformation efficiencies

The effect of the incubation time between the buffer cells transformation.




Incubation Time
Heat Shock Step

After 15 min incubation


efficiency = 0.8 x 10^4 transformants per µg of DNA CFU/mL = 0.8 x 10^4

After 30 min incubation


Transformation efficiency = 1.64 x 10^4 transformants per µg of DNA CFU/mL = 1.64 x 10^4


Comment


The results show a doubled transformation efficiencies when cells were incubated with the buffer 30mins compared to those incubated for 15min.


The effect of heat-shock step on the transformation


a)Transformation with heat-shock


- No. of colonies = 150
- Vol. of culture plated = 100µl
- Transformation efficiency = 1.5 x 10^4 transformants per µg
- CFU/mL = 3 x 10^4

Transformation without heat-shock



- No. of colonies = 133
- Vol. of culture plated = 100µl
- Transformation efficiency = 1.33 x 10^4 transformants per µg
- CFU/mL = 2.66 x 10^4

Comment


The results show very close transformation efficiency values, with slightly higher value for cells that were heat-shocked. This confirms that the buffer can work without the need for heat-shock.

Summary

We recommend you work with TSS buffer while preparing competent cells for the highest transformation efficiency incubate the buffer with cells for 30 mins, the buffer also works fine without a recovery step and a heat-shock step can be also neglected and get considerably same efficiency for the transformation, but it is recommended to heat-shock cells to achieve the highest efficiency.

Miniprep optimization


Different protocols were tested on BL21 for the highest yield and purity of plasmid DNA
Lithium method
TELT method
Phenol/chloroform method
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Protocol overview


Plasmid DNA is extracted from E. coli colonies grown on plates or in liquid cultures. Bacterial cells containing plasmid DNA are treated with Triton X-100/LiCl and phenol/chloroform in that order. These steps dissolve plasmid DNA while precipitating chromosomal DNA in the presence of cellular debris. Centrifugation is used to remove the debris. This isolation method produces plasmid DNA preparations that are nearly devoid of chromosomal DNA.


Protocol link

Results


Spectroscopic analysis:

Conc. = 0.43 µg/µl
Purity = 2.77

Protocol link

Results


Spectroscopic analysis:

Conc. =0.39 µg/µl
Purity = 2.77

Protocol overview


Mini-Prep procedure is used to isolate small plasmid DNA from bacteria while limiting contaminating proteins and genomic DNA; it relies on the principle of liquid-liquid extraction of biomolecules. It denatures the protein part and separates the genomic DNA into a soluble phase.

Protocol link

Results


Spectroscopic analysis:

Conc. = 1.5 µg/µl
Purity = 1.1

DNA-gel electrophoresis for the samples


1; 1Kb DNA ladder, 3; Lithium method miniprep, 7-8; TELT method, 9; Phenol/chloroform method

Comment

From spectrophotometric data and gel results, phenol/chloroform method was our best option due to the high DNA contamination yielded from TELT method, and the low plasmid concentration yielded from the `lithium method.

The effect of the bacterial strain on the plasmid yielded from the miniprep reaction and concentration

pGS-21a was minipreped from E. coli DH5-alpha & BL21 (DE3) using phenol/chloroform method

pGS-21a from DH5-alpha
pGS-21a from BL21(DE3)
Results

DNA-gel electrophoresis results:


1; 1Kb DNA ladder, 3; pGS-21a miniprep from 1.5ml DH5-alpha, 5;


Comment


Bands intensities referred to higher minipreped plasmids from a culture of 1.5mL that of 3mL

DNA-gel electrophoresis results


1; 1Kb DNA ladder, 3; Lithium method miniprep, 7-8; TELT method, 9; Phenol/chloroform method


Comment


The plasmid band resulted from DH5-alpha, and the calculated spectrophotometric concentration confirms the fact that DH5-alpha is a better chassis to store your plasmid and miniprep them.

Ligation Reaction


We’ve tested the effect of the incubation time and the insert: vector ratio on the ligation efficiency on blunt-ended and sticky ended inserts and vector.
Blunt ended

Tested Conditions


1) At 25 °C for 3 hrs. (3:1)
2) At 25 °C for 18 hrs. (3:1)
3) At 15 °C for 18 hrs. (3:1)




The best Conditions from the tested ones


1) At 15 °C for 18 hrs. (3:1)
results of agarose gel electrophoresis after ligation in the blunt-ended vector (pJET).



The gel image indicates that we had successful ligation with our optimized condition between the (pJET) vector and our parts. We could confirm the ligation as we had a successful Transformation.

Restriction digestion


We’ve optimized a protocol to work with two different enzymes with their separate buffers, in our case we used two restriction enzymes from Thermo Scientific™, the first was XbaI (cat. No: ER0681), while the second was FastDigest XhoI (cat. No: FD0694)

Double digest

In the double digest the two enzymes are used together at the same time.



Result

no restriction achieved

Sequential Restriction

A sequential digest should be employed if there is no buffer in which the two enzymes demonstrate >50% activity. The procedure starts by using the restriction enzyme with the lowest salt concentration. Two incubation conditions were tested the first XbaI was incubated for 7hr while XhoI was incubated for 15min (protocol 1), the alternative conditions were incubating XbaI for 8hr while XhoI for 30min (protocol 2).


Result


lane1; 1Kb DNA ladder, lane7; restriction digestion using “protocol 2”, lane8; restriction digestion using “protocol 1”

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The best conditions that totally restricted the sample was the conditions provided in “protocol 2”

Protein expression


Optimizations for the cell lysis method

Bacterial cells can be lysed by physical lysis methods using sonication or chemical lysis using chemical buffers, we’ve tested the effect of those methods on the protein yield. All proteins were lysed from BL21(DE3) cells and their concentrations were characterized using BSA assay.

BSA results

Protein name

Soniation Protein concentration (mg/mL)

Chemical Lysis Protein concentration (mg/mL)

His-Tau (ID: BBa_K4165009)

0.016

0.3277

GST-linker-CoH2-WWW (ID: BBa_K4165256)

0.0246

0.7195

GST-linker-CoH2- TD28REV (ID: BBa_K4165255)

0.051

0.4976

GST-DoCS (16h) (ID: BBa_K4165253)

0.0664

0.8289

His-CoH2 (ID: BBa_K4165003)

-0.0287

0.2867

GST-CoH2 (ID: BBa_K4165254)

1.241

0.1158

GST-DoCS(3h) (ID: BBa_K4165253)

-0.2078

-

His-TRIM21-linker- DoCS (ID: BBa_K4165202)

0.5723

0.2858

His-DoCS (3h) (ID: BBa_K3396000)

-0.4714

-

His-DoCS (16h) (ID: BBa_K3396000)

-0.8335

0.8574

Comment


Chemical lysis enhanced the protein yield for all the parts significantly except for the GST-CoH2


Protocols


1) All the sonication conditions for each part are provided in the experiments section
2) Chemical lysis protocol:

Optimizations for Dockerin expression

⮚ The effect of the induction time and type of tag on the yield of protein

Yield after 3h induction
Yield 16h induction
Summary

1. GST-tagged

BCA assay result


Concentration from sonication lysis = -0.207mg/mL.
Comment: the negative results can be resulted from an insufficient lysis conditions, low IPTG concentration or a problem with the induction time.

2.His-tagged

BCA assay result

Concentration from sonication lysis = -0.47mg/mL.
Comment: the negative results can be resulted from an insufficient lysis conditions, low IPTG concentration or a problem with the

1. GST-tagged:

BCA assay result


Concentration from sonication lysis = 0.0664 mg/mL.
Concentration from chemical lysis = 0.8289 mg/mL.
Comment: Data shows an increase in the protein yield using chemical lysis compared to sonication method.

2.His-tagged

BCA assay result


Concentration from sonication lysis = -0.834 mg/mL.
Concentration from chemical lysis = 0.8574

Comment Data shows an increase it the protein yield using chemical lysis compared to sonication method. the negative results can be resulted from an insufficient lysis conditions, low IPTG concentration or a problem with the induction time.

Summary


Using sonication lysis method; after 3hr induction no protein yield was seen in both tags, while after 16hr induction GST-tagged dockerins yielded a considerably low concentration, while his-tagged dockerins showed no yield at all. Using chemical lysis; after 16hr induction GST-tagged Dockerins yielded a high protein concentration compared to His-tagged Dockerins which confirms that GST increased the domain yield

Optimizations for Cohesin expression

The effect of the tag type on the protein yield

GST-tagged CoH2
His-tagged CoH2
Summary

BCA assay result


Concentration from sonication lysis = 1.2405 mg/mL.
Concentration from chemical lysis = 0.1158 mg/mL

Comment

unlike other proteins; sonication significantly increased the yield of the protein compared to chemical lysis

BCA assay result


Concentration from sonication lysis = -0.0287 mg/mL.
Concentration from chemical lysis = 0.2867 mg/mL

Comment


Data shows a significant increase in the protein yield using chemical lysis compared to sonication method. the negative results can be resulted from an insufficient lysis conditions, low IPTG concentration or a problem with the induction

Summary


Using sonication lysis method; after 3hr induction a high protein yield was observed in GST-tagged domains, while no yield was observed for the his-tagged domains under the same conditions. On the other hand, chemical lysis surprisingly decreased the GST- tagged domains yield, while maintained a conventional yield for the his-tagged ones compared with sonication that gave no yield.