Molecular Biology ex­per­i­ments #

Protocols for Cloning

PROTOCOLS FOR THE EXPERIMENT PERFORMED

E.coli RELATED PROTOCOL #

Preparation of buffers and Media #

1. LB Medium
2. We used ready­made LB agar and Broth(Himedia - Broth and Agar). For broth - add 40 grams per 1000 ml of wa­ter.
3. For agar - add 25 grams per 1000 ml of wa­ter.Ster­il­ize these so­lu­tions by au­to­clav­ing at 15 lbs at $121\mathrm{°}C$ for 15 min­utes.

E.coli com­pe­tent cell prepa­ra­tion #

1. Competent cells prepa­ra­tion by Inoue method - used to pre­pare DH5α (Adapted from https://​bio-pro­to­col.org/​ex­change/​pro­to­cold­e­tail?id=143&type=1)

• A sin­gle bac­te­r­ial colony was picked up from a plate and in­cu­bated in a 100 ml cul­ture tube overnight in a shaker in­cu­ba­tor(250-300 rpm, $37\mathrm{°}C$).This is the Primary cul­ture.
• Take this pri­mary cul­ture and pour it into fresh LB Medium - split in three cul­ture tubes. Put in 10 ml of this cul­ture in the first cul­ture tube, 4ml in the sec­ond cul­ture tube and 2ml cul­ture medium in the third one. Incubate these flasks overnight at $18-22\mathrm{°}C$. This is the sec­ondary cul­ture. Setting up three cul­tures helps ac­count for the vari­ables in lab con­di­tions - which may cause cul­tur­ing rates to vary.
• When the O.D of one of the cul­tures reaches 0.55, trans­fer the cul­ture into an ice bath.Dis­card the other two cul­tures. Centrifuge this for $2500\times g$ for 10 min at $4\mathrm{°}C$ to har­vest the cells
• Pour off the medium thor­oughly - in­vert the tube over a cou­ple of pa­per tow­els in or­der to dab out any ex­tra medium. You could also use a vac­uum as­pi­ra­tor in or­der to re­move any medium stuck to the walls.
• Resuspend the cells us­ing an 80ml Inoue trans­for­ma­tion buffer. Centrifuge this for $2500\times g$ for 10 min at $4\mathrm{°}C$ to har­vest the cells. Repeat pre­vi­ous step.Com­pe­tent cells are ready.Now pre­pare them for stor­age.

Cold stor­age of com­pe­tent cells

• Resuspend the cells us­ing a 20 ml Inoue Transformation buffer.Add 1.5 ml of DMSO, mix the bac­te­r­ial sus­pen­sion and store in ice.
• Aliquot the con­tent into a num­ber of mi­cro­cen­trifuge tubes (MCT chilled by stor­ing in ice). Now flash freeze these MCTs con­tain­ing com­pe­tent cells us­ing liq­uid ni­tro­gen.
• Freeze these tubes at $-70\mathrm{°}C$.

Inoue trans­for­ma­tion buffer #

Reagent	Amount/L	Final Concentration
$MnC{l}_2$ $4H_{2}O$	10.88 g	55mM
$CaC{l}_2$ $2H_{2}O$	2.20 g	15 mM
$KCl$	18.65 g	250 mM
PIPES (0.5 M, pH 6.7)	20 ml	10 mM
$H_{2}O$	to 1 L

PIPES Solution - Prepare a 0.5 M stock so­lu­tion by dis­solv­ing 15.1 g PIPES to 80ml pure wa­ter.

Transformation pro­to­col for E.coli #

Thaw one vial of the com­pe­tent cells and add up to 25 ng per 50 ml of plas­mid DNA.Swirl the tube a few times.The ap­pro­pri­ate pos­i­tive and neg­a­tive con­trols per ex­per­i­ment was en­sured dur­ing the ex­per­i­ment con­trols.

These cells were then given a heat shock at $42\mathrm{°}C$ in a wa­ter bath for 45-50secs fol­lowed by im­me­di­ate trans­fer to ice for 1-2mins.

1. 800 ml of SOC medium is added to each tube fol­low­ing which the tubes are trans­ferred into a shaker in­cu­ba­tor set at $37\mathrm{°}C$ for 45 min­utes.
2. These trans­formed bac­te­ria are spread on a plate with ap­pro­pri­ate an­tibi­otics to se­lect for trans­formed cells over non-trans­formed cells. For ampi­cillin re­sis­tant plates -

• Don’t in­cu­bate the plates for more than 20 hours at $37\mathrm{°}C$.
• Cell den­sity must be kept low - Less than 104 cells per 90mm.Sometimes, the re­lease of β-lac­ta­mase, which re­sults in the degra­da­tion of ampi­cillin, could form satel­lite colonies which are sen­si­tive to ampi­cillin - re­sult­ing in in­cor­po­ra­tion of non-trans­formed cells. Using car­beni­cillin in place of ampi­cillin ame­lio­rates this.

For tetra­cy­cline plates You can plate the whole trans­for­ma­tion mix­ture.For this, har­vest the bac­te­ria by cen­trifu­ga­tion for 20 sec­onds and re­sus­pend the bac­te­ria in 100 ml medium(LB).

D.PCR Protocols During the course of our pro­ject, we used the fol­low­ing en­zymes -

1. NEB Q5 DNA Polymerase
2. Thermo Fisher Scientific Phusion High fi­delity DNA Polymerase
   • Error rate is 50 fold lower than the tra­di­tional Taq poly­merase.
   • Highly pro­ces­sive - which makes it a bet­ter choice for cloning.
3. Takara rTaq DNA Polymerase

Da.PCR Reaction mix­ture for phu­sion:

Component	20 $\mu L$ rxn	50 $\mu L$ rxn	Final Conc.
$H_{2}O$	add to 20 $\mu L$	add to 50 $\mu L$	-
5X PhusionTM Buffer	4$\mu L$	10 $\mu L$	1X
10 mM dNTP	0.4 $\mu L$	1 $\mu L$	200 $\mu L$ each
Forward Primer	X $\mu L$	X $\mu L$	0.5 $\mu M$
Reverse Primer	X $\mu L$	X $\mu L$	0.5 $\mu M$
Template DNA	X $\mu L$	X $\mu L$	-
DMSO, Optional	0.6 $\mu L$	1.5 $\mu L$	3%
PhusionTM High-Fidelity DNA Polymerase	0.2 $\mu L$	0.5 $\mu L$	0.02 U/$\mu L$

Db. PCR re­ac­tion for q5:

Component	25 $\mu L$ Reaction	50 $\mu L$ Reaction	Final Concentration
5XQ5 Reaction Buffer	5 $\mu L$	10 $\mu L$	1X
10 mMd­NTPs	0.5 $\mu L$	1 $\mu L$	200 $\mu M$
10 $\mu M$ Forward Primer	1.25 $\mu L$	2.5 $\mu L$	2.5 $\mu L$
10 $\mu M$ Reverse Primer	1.25 $\mu L$	2.5 $\mu L$	0.5 $\mu L$
Template DNA	Variable	Variable	< 1,000 ng
Q5 High Fidelity DNA Polymerase	0.25 $\mu L$	0.5 $\mu L$	0.02 U/$\mu L$
5XQ5 HighGCEnhancer (Optional)	5 $\mu L$	10 $\mu L$	1X
Nuclease - Free Water	to 25 $\mu L$	to 50 $\mu L$	-

Dc.PCR Protocol for Taq DNA Polymerase:

Component	25 $\mu L$ Reaction	50 $\mu L$ Reaction	Final Concentration
10X Standard Taq Reaction Buffer	2.5 $\mu L$	5 $\mu L$	1X
10 mMd­NTPs	0.5 $\mu L$	1 $\mu L$	200 $\mu M$
10 $\mu M$ Forward Primer	0.5 $\mu L$	1 $\mu L$	0.2 $\mu M$ (0.05–1 $\mu M$)
10 $\mu M$ Reverse Primer	0.5 $\mu L$	1 $\mu L$	0.2 $\mu M$ (0.05–1 $\mu M$)
Template DNA	Variable	Variable	< 1,000 ng
Taq DNA Polymerase	0.125 $\mu L$	0.25 $\mu L$	1.25 units/​50 $\mu L$ PCR
Nuclease - Free Water	to 25 $\mu L$	to 50 $\mu L$	-

E.Plasmid Extraction Protocol #

We are us­ing Favorgen FavorPrep™ Plasmid DNA Extraction Mini Kit (Catalog no : FAPDE 300) for E.coli Plasmid Extraction.

DAY - 1

1. Pick a sin­gle bac­te­r­ial colony from the plas­mid-con­tain­ing bac­te­r­ial plate and in­oc­u­late it in 5ml LB Broth in 55ml Culture Tubes with the ap­pro­pri­ate se­lec­tion an­tibi­otic.
2. Let the cul­ture grow for 12-16 hours on $37\mathrm{°}C$ with 200 rpm shak­ing.

DAY - 2

1. Transfer 2ml of well-grown bac­te­r­ial cul­ture to a 2ml mi­cro­cen­trifuge tube.
2. Centrifuge the tube at $11,000\times g$ for 1 minute to pel­let the cells and dis­card the su­per­natant com­pletely.
3. Repeat the first step till all the 5ml bac­te­r­ial cul­ture has been pel­leted down.
4. Add 200 $\mu L$ of FAPD1 Buffer (RNase A added) to the cell pel­let and re­sus­pend the cells com­pletely by pipet­ting.
   • Make sure that RNase A has been added into the FAPD1 Buffer when first used.
   • No cell pel­let should be vis­i­ble af­ter the re­sus­pen­sion of the cells.
5. Add 200 $\mu L$ of FAPD2 Buffer and gen­tly in­vert the tube 8 ~ 10 times. Incubate the sam­ple mix­ture at room tem­per­a­ture for 4 ~ 5 min­utes to lyse the cells.
   • Do not vor­tex; vor­tex­ing may shear ge­nomic DNA. If nec­es­sary, con­tinue in­vert­ing the tube un­til the lysate be­comes clear.
   • Do not pro­ceed with the in­cu­ba­tion for over 5 min­utes.
6. Add 300 $\mu L$ of FAPD3 Buffer and in­vert the tube 5 ~ 10 times im­me­di­ately to neu­tral­ize the lysate.
   • Inverting im­me­di­ately af­ter adding FAPD3 Buffer will avoid asym­met­ric pre­cip­i­ta­tion.
7. Centrifuge at full speed (~18,000 x g) for 7 min to clar­ify the lysate.
8. During cen­trifu­ga­tion, place a FAPD Column in a Collection Tube.
9. Transfer the su­per­natant care­fully to the FAPD Column and cen­trifuge at 11,000 x g for 1 minute.Dis­card the flow-through and place the col­umn back to the Collection Tube.
   • Do not trans­fer any white pel­let into the col­umn.
10. Add 400 $\mu L$ of WP Buffer to the FAPD Column and cen­trifuge at 11,000 x g for 1 minute.Dis­card the flow-through and place the col­umn back in the Collection Tube.
11. Add 700 $\mu L$ of Wash Buffer to the FAPD Column and cen­trifuge at 11,000 x g for 1 minute.Dis­card the flow-through and place the col­umn back to the Collection Tube.
    • Make sure that ethanol (96-100 %) has been added into the Wash Buffer when first used.
12. Centrifuge at full speed (~ 18,000 x g) for an ad­di­tional 5 min­utes to dry the FAPD Column.
    • The resid­ual liq­uid should be re­moved thor­oughly on this step.
13. Place the FAPD Column into a new 1.5 ml mi­cro­cen­trifuge tube.
14. Warm dd$H_{2}O$ by plac­ing it into a dry bath at $65\mathrm{°}C$
15. Add 30 $\mu L$ of the warm dd$H_{2}O$ to the mem­brane cen­ter of the FAPD Column.Stand the col­umn for 3 min­utes.
    • For ef­fec­tive elu­tion, make sure that the wa­ter is dis­pensed on the mem­brane cen­ter and is ab­sorbed com­pletely.
16. Centrifuge at full speed (~ 18,000 x g) for 2 min­utes to elute plas­mid DNA
17. Repeat the 15th Step by adding the eluted plas­mid once again to the mem­brane cen­ter of the FAPD Column.
18. Centrifuge fi­nally at full speed (~ 18,000 x g) for 2 min­utes to get the plas­mid DNA in the mi­cro­cen­trifuge.
19. Store the plas­mid DNA at -20 °C.

The above pro­to­col is the op­ti­mized pro­to­col used by our team.The stan­dard kit pro­to­col can be found here.

Restriction di­ges­tion pro­to­col #

Restriction Enzyme	10 units is Sufficient, gen­er­ally 1 $\mu L$ is used
DNA	1 µg
10X NEBuffer	5 $\mu L$ (1X)
Total Reaction Volume	50 $\mu L$
Incubation Time	1 Hour
Incubation Temperature	Enzyme Dependent

Buffer

• Use at a 1X con­cen­tra­tion Sup­ple­ment with SAM (S-Adenosylmethionine) to the rec­om­mended con­cen­tra­tion if re­quired.

Reaction Volume

• A 50 $\mu L$ re­ac­tion vol­ume is rec­om­mended for di­ges­tion of 1 µg of sub­strate

Incubation Time

• Incubation time is typ­i­cally 1 hour

Storage

• Storage at -20°C is rec­om­mended for most re­stric­tion en­zymes

Gel DNA Extraction Protocol #

We are us­ing Favorgen FavorPrepTM GEL/ PCR Purification Kit(Catalogue no :- FAGCK 001-1) for ex­tract­ing DNA frag­ments from the agarose gel.

1. Excise the agarose gel with a clean scalpel.

   • Remove the ex­tra agarose gel to min­i­mize the size of the gel slice.

2. Transfer up to 300 mg of the gel slice into a mi­cro­cen­trifuge tube.

3. Add 500 $\mu L$ of FADF Buffer to the sam­ple and mix by vor­tex­ing. For > 2% agarose gels, add 1000 $\mu L$ of FADF Buffer.

4. Incubate at 55 °C for 20~30 min­utes and vor­tex the tube every 5 min­utes un­til the gel slice dis­solved com­pletely.

   • During in­cu­ba­tion, in­ter­val vor­tex­ing can ac­cel­er­ate the gel dis­solved.
   • Make sure that the gel slice has been dis­solved com­pletely be­fore pro­ceed­ing to the next step.
   • After the gel dis­solved, make sure that the color of the sam­ple mix­ture is yel­low. If the color is vi­o­let, add 10 $\mu L$ of sodium ac­etate, 3M,pH 5.0.Mix well to make the color of the sam­ple mix­ture turn to yel­low.

5. Cool down the sam­ple mix­ture to room tem­per­a­ture.And place a FADF Column into a Collection Tube.

6. Transfer 800 $\mu L$ of the sam­ple mix­ture to the FADF Column.Centrifuge at 11,000 x g for 1 minute, then dis­card the flow-through.

   • If the sam­ple mix­ture is more than 800 $\mu L$, re­peat this step for the rest of the sam­ple mix­ture.

7. Add 750 $\mu L$ of Wash Buffer (ethanol added) to the FADF Column.Centrifuge at 11,000 x g for 1 minute, then dis­card the flow-through.

   • Make sure that ethanol (96-100 %) has been added into the Wash Buffer when first used.

8. Repeat the above step once again.

9. Centrifuge at full speed (~ 18,000 x g) for an ad­di­tional 5 min­utes to dry the col­umn ma­trix.

• Important step ! The resid­ual liq­uid should be re­moved thor­oughly on this step.

10. Place the FADF Column to a new mi­cro­cen­trifuge tube.
11. Warm dd$H_{2}O$ by plac­ing it into a dry bath at $65\mathrm{°}C$.
12. Add 30 $\mu L$ of the warm dd$H_{2}O$ to the mem­brane cen­ter of the FAPD Column.Stand the col­umn for 3 min­utes.

• For ef­fec­tive elu­tion, make sure that the wa­ter is dis­pensed on the mem­brane cen­ter and is ab­sorbed com­pletely.

13. Centrifuge at full speed (~ 18,000 x g) for 2 min­utes to elute the DNA
14. Repeat the 12th Step by adding the eluted DNA once again to the mem­brane cen­ter of the FAPD Column.
15. Centrifuge fi­nally at full speed (~ 18,000 x g) for 2 min­utes to get the eluted DNA in the mi­cro­cen­trifuge.
16. Store the DNA at -20 °C.

The above pro­to­col is the op­ti­mized pro­to­col used by our team.The stan­dard kit pro­to­col can be found here.

PCR RELATED PROTOCOL #

PCR / Reaction Mixture Cleanup Protocol #

We are us­ing Favorgen FavorPrepTM GEL/ PCR Purification Kit(Catalogue no :- FAGCK 001-1) to pu­rify PCR prod­ucts of re­ac­tion mix­tures(for con­cen­tra­tion and de­sali­na­tion of re­ac­tion mix­tures)

1. Transfer up to 100 $\mu L$ of PCR prod­uct (excluding oil) to a mi­cro­cen­trifuge tube and add 5 vol­umes of FADF Buffer, mix well by vor­tex­ing and spin it down.
   • For ex­am­ple, Add 250 $\mu L$ of FADF Buffer to 50 $\mu L$ of PCR prod­uct. The max­i­mum vol­ume of PCR prod­uct is 100 $\mu L$ (excluding oil).Do not ex­ceed this limit. If the PCR prod­uct is more than 100 $\mu L$,separate it into mul­ti­ple tubes.
2. Place a FADF col­umn into a Collection Tube.
3. Transfer the sam­ple mix­ture to the FADF Column.Centrifuge at 11,000 x g for 1 minute, then dis­card the flow-through.
4. Add 750 $\mu L$ of Wash Buffer (ethanol added) to the FADF Column.Centrifuge at 11,000 x g for 1 minute, then dis­card the flow-through.
   • Make sure that ethanol (96-100 %) has been added into the Wash Buffer when first used.
5. Repeat the above step once again.
6. Centrifuge at full speed (~ 18,000 x g) for an ad­di­tional 5 min­utes to dry the col­umn ma­trix.
   • The resid­ual liq­uid should be re­moved thor­oughly on this step.
7. Place the FADF Column to a new mi­cro­cen­trifuge tube.
8. Warm dd$H_{2}O$ by plac­ing it into a dry bath at $65\mathrm{°}C$
9. Add 30 $\mu L$ of the warm dd$H_{2}O$ to the mem­brane cen­ter of the FAPD Column.Stand the col­umn for 3 min­utes.
   • For ef­fec­tive ex­trac­tion, make sure that the wa­ter is dis­pensed on the mem­brane cen­ter and is ab­sorbed com­pletely.
10. Centrifuge at full speed (~ 18,000 x g) for 2 min­utes to elute the DNA
11. Repeat the 9th Step by adding the eluted DNA once again to the mem­brane cen­ter of the FAPD Column.
12. Centrifuge fi­nally at full speed (~ 18,000 x g) for 2 min­utes to get the eluted DNA in the mi­cro­cen­trifuge.
13. Store the DNA at -20 °C.

The above pro­to­col is the op­ti­mized pro­to­col used by our team. The stan­dard kit pro­to­col can be found here.

Ligation Protocol #

We are us­ing NEB T4 DNA lig­ase (Catalogue No:- M0202) for lig­a­tion re­ac­tions.

Set up the fol­low­ing re­ac­tion in a mi­cro­cen­trifuge tube on ice.

COMPONENT	20 $\mu L$ REACTION
T4 DNA Ligase Buffer (10X)	2 $\mu L$
Vector DNA (4 kb)	50 ng (0.020 pmol)
Insert DNA (1 kb)	37.5 ng (0.060 pmol)
dd$H_{2}O$	to 20 $\mu L$
T4 DNA Ligase	1 $\mu L$

T4 DNA Ligase should be added last.Note that the table shows a lig­a­tion us­ing a mo­lar ra­tio of 1:3 vec­tor to in­sert for the in­di­cated DNA sizes.) Use NEBioCalculator to cal­cu­late mo­lar ra­tios for your lig­a­tion re­ac­tion

1. The T4 DNA Ligase Buffer should be thawed and re­sus­pended at room tem­per­a­ture.
2. Gently mix the re­ac­tion by pipet­ting up and down and mi­crofuge briefly.
3. For co­he­sive ends, in­cu­bate at 16°C for about 12-16 hours.
4. For blunt ends or sin­gle base over­hangs, in­cu­bate at $16\mathrm{°}C$ overnight
5. Heat in­ac­ti­vated at 65°C for 10 min­utes.
6. Chill on ice and trans­form 10 $\mu L$ of the re­ac­tion into 100 $\mu L$ com­pe­tent cells

COLONY PCR #

cPCR Protocol us­ing Taq DNA Polymerase:

Component	25 $\mu L$ Reaction	50 $\mu L$ Reaction	Final Concentration
10X Standard Taq Reaction Buffer	2.5 $\mu L$	5 $\mu L$	1X
10 mMd­NTPs	0.5 $\mu L$	1 $\mu L$	200 $\mu M$
10 $\mu M$ Forward Primer	0.5 $\mu L$	1 $\mu L$	0.2 $\mu M$ (0.05–1 $\mu M$)
10 $\mu M$ Reverse Primer	0.5 $\mu L$	1 $\mu L$	0.2 $\mu M$ (0.05–1 $\mu M$)
Template DNA	Variable	Variable	< 1,000 ng
Taq DNA Polymerase	0.125 $\mu L$	0.25 $\mu L$	1.25 units/​50 $\mu L$ PCR
Nuclease - Free Water	to 25 $\mu L$	to 50 $\mu L$	-

Bacillus sub­tilis RELATED PROTOCOL #

BIOLOGY AND GENETICS OF Bacillus sub­tilis #

Microbiology of Bacillus sub­tilis

The model or­gan­ism which we are us­ing in our pro­ject is a gram-pos­i­tive bac­te­ria, B.subtilis. Its rod-shaped cells typ­i­cally range in length from 4 to 10 m, and their di­am­e­ter is be­tween 0.25 and 1 m. Their colonies are ovoid, opaque, smooth, off-white, and slightly raised. It can pro­duce en­dospores in an un­fa­vor­able en­vi­ron­ment and thus en­dure un­til its cir­cum­stances im­prove. It can be dis­cov­ered in soil and in some mam­mals’ gas­troin­testi­nal tracts, in­clud­ing hu­mans. In mol­e­c­u­lar bi­ol­ogy and biotech­nolo­gies, B.subtilis is fre­quently used. It is re­garded as the Gram-positive ver­sion of Escherichia coli be­cause of the wealth of knowl­edge and ge­netic re­sources avail­able.The safety of B.subtilis is also ac­knowl­edged (GRAS)

Phylogenesis and na­ture habi­tat of Bacillus sub­tilis

B.subtilis is a mem­ber of the genus Bacillus, which also con­tains well-known species like Bacillus cereus, B.licheniformis (used in the pro­duc­tion of an­tibi­otics), and B.thuringiensis (used for pro­duc­tion of spe­cific in­sec­ti­cides).Most mem­bers of this genus are non-path­o­genic or low path­o­genic, with the ex­cep­tion of B.anthracis.The fam­i­lies Staphylococcaceae and Bacillaceae are re­lated be­cause they are both mem­bers of the class Bacilli.Bacilli and Clostridia make up the low GC class of Gram-positive bac­te­ria known as fir­mi­cutes. You can ac­cess its en­tire tax­on­omy here: http://​lifemap.univ-ly­on1.fr/​ex­plore.html

Gram-positive B.subtilis bac­te­ria can pro­duce ex­tra­cel­lu­lar pro­teins be­cause they lack an outer mem­brane.B.subtilis is there­fore very use­ful in biotech­nol­ogy.Gram-pos­i­tive bac­te­ria need ex­tra pro­tec­tion if their outer mem­brane is miss­ing in or­der to pre­vent harm.For in­stance, they are re­sis­tant to lysozymes due to mod­i­fi­ca­tions to their cell walls.B.subtilis is fre­quently found in up­per soil lay­ers and in the gas­troin­testi­nal tract of some mam­mals, in­clud­ing hu­mans.The ideal growth con­di­tions for B.subtilis, a strictly aer­o­bic or­gan­ism, are be­tween 30 and 37 °C, with a min­i­mum tem­per­a­ture of 18 °C and a max­i­mum tem­per­a­ture of 43 °C.The for­ma­tion of en­dospores is typ­i­cal of the genus Bacillus. Internal com­part­ments de­velop in starved cells and later de­velop into en­dospores.These en­dospores are re­silient.

The dor­mant cells re­ac­ti­vate into veg­e­ta­tive states through ger­mi­na­tion.The trig­gers for this are fre­quently amino acids and pep­ti­do­gly­can muropep­tides, which are re­leased by de­vel­op­ing cells. In spe­cific cir­cum­stances, B.subtilis is able to form biofilms.A bi­olm is a struc­ture used by bac­te­ria for ad­he­sion to sur­faces, com­mu­ni­ca­tion, and self-de­fense.Where bi­olm is pri­mar­ily cre­ated is at the air-liq­uid in­ter­face.Bac­te­ria are pro­duced as a re­sult of dis­solved oxy­gen de­ple­tion and ad­sorp­tion.Bi­olm is ad­van­ta­geous for the sur­vival of the colonies be­cause it can pro­tect them from pro­to­zoal preda­tors, chang­ing cli­matic con­di­tions, nu­tri­ent de­ple­tion, and un­fa­vor­able pH.Pro­teases, par­tic­u­larly sub­tilis in and neu­tral pro­tease (nPro), which can af­fect the pro­duc­tion of re­com­bi­nant pro­teins, may com­pli­cate the use of B.subtilis in in­dus­try.How­ever, there are nu­mer­ous strains that have dele­tions in the genes re­spon­si­ble for pro­duc­ing these en­zymes.The well-known B.subtilis WB800 strain, which has 8 key ex­tra­cel­lu­lar pro­teases deleted, is an il­lus­tra­tion of such a strain.These en­zymes are also es­sen­tial for the de­vel­op­ment of bac­te­ria.

Genetics of Bacillus sub­tilis

The first Gram-positive bac­terium with a known genome se­quence was Bacillus sub­tilis, which was se­quenced in 1997.A to­tal of 1500 oper­ons are made up of about 4000 genes.A sig­nif­i­cant por­tion of the re­main­ing genome is used for growth and sur­vival in the en­vi­ron­ment, while slightly more than half of the genome is nec­es­sary for cell processes, in­ter­me­di­ary me­tab­o­lism, and macro­mol­e­c­u­lar syn­the­sis.Nat­ural trans-for­ma­tion is the method most fre­quently used to in­tro­duce iso­lated DNA into B.subtilis.Recombining im­ported DNA with its chro­mo­so­mal ho­mol­o­gous se­quences is a very ef­fi­cient process. It is pos­si­ble to ex­press the de­sired gene di­rectly from a plas­mid, though this method typ­i­cally has a lower suc­cess rate than chro­mo­so­mal in­te­gra­tion.

The ex­pla­na­tion is straight­for­ward: During en­try, vec­tors are trans­formed into a sin­gle-stranded state and ran­domly frag­mented.Since in­te­gra­tion plas­mids do not need to con­tinue to be fully func­tional and self-repli­cat­ing, this is not a prob­lem. By in­te­grat­ing at the cho­sen in­ser­tion site, the per­ti­nent genes are “rescued.” Since the AmyE test can be used to de­ter­mine whether the in­te­gra­tion was suc­cess­ful, the AmyE site, cod­ing -amylase, is fre­quently used.

How to cul­ti­vate?

B.subtilis is very easy to cul­ti­vate.We use the same cul­ti­va­tion con­di­tions as for E.coli.The overnight cul­tures grow with no is­sues at 37 °C and 200 rpm.We usu­ally use 50 ml fal­cons with 10 ml of LB medium.The larger vol­ume of the flask is bet­ter for prop­per shak­ing. It is bet­ter to lay the flask on its side for bet­ter move­ment of the cul­ture.Al­though B.subtilis grows well also in lower tem­per­a­ture, the lab­o­ra­tory rou­tine is to cul­ti­vate it in 37 °C (in uid medium and also on plates) - colonies are larger and it takes less time. If you want to slow down the growth, you can leave plates at room tem­per­a­ture (e.g.for the week­end), but make sure that the UV light is not used to dis­in­fect your lab­o­ra­tory overnight.Let the colonies grow up­side down on the plate.

How to store Bacillus sub­tilis?

The best way of stor­ing B.subtilis, as well as E.coli, cul­tures is as glyc­erol stocks.Use LB agar to densely spread one colony of B.subtilis (use ster­ile mi­cro­bi­o­log­i­cal loop) on the plate.Fol­low­ing day, har­vest the plates and trans­port the bac­te­ria into 2 ml of liq­uid LB medium with 20% glyc­erol (sterile).These glyc­erol stocks are then stored at -80 °C.You could also use HMFM for prepar­ing stocks. If you need to use a sam­ple from the stock, put it out of the freezer, open it on ice in a low box and sim­ply scoop up a small amount of frozen cul­ture in the me­dia or on the top of the agar plate with a ster­ile loop.Work quickly and do not let the stocks melt.Af­ter­wards, you can close the stock and re­turn it to -80 °C.

Which an­tibi­otics could be used for se­lec­tion of trans­for­mants?

Be cau­tious! Ampicillin has no ef­fect on B.subtilis.Avoid at­tempt­ing to se­lect it us­ing these an­tibi­otics. It is not func­tional (our own ex­pe­ri­ence).Shut­tle vec­tors, which are also used with E.coli, are most likely those that are suit­able for B.subtilis and also con­tain the bla gene.The se­lec­tion of an an­tibi­otic is based on the avail­able se­lec­tion sys­tem, which is fre­quently in­flu­enced by the re­sis­tance gene found in the vec­tor’s se­quence.We used pDG vec­tros, which code for ery­thromycin, spectin­o­mycin, and chlo­ram­pheni­col re­sis­tance, in our cam­paign.Ery­thromycin works best when com­bined with lin­comycin (also known as MLS se­lec­tion) be­cause there are never as many colonies of the neg­a­tive con­trol af­ter trans­for­ma­tion.

ATB	Usage for:	Vector	Stock Concentration	Dilution in	Final con­cen­tra­tion
Ampicillin	se­lec­tion of trans­for­mants in E. coli	pDG3661 pDG1664 pB­S1C pB­S2E	150 mg/​ml	wa­ter	100 μg/​ml
Erythromycin	se­lec­tion of trans­for­mants in B.subtilis , in comb. with lin­comycin	pDG1664	10 mg/​ml	ethanol	0.5 μg/​ml*
Lincomycin	se­lec­tion of trans­for­mants in B.subtilis , in comb. with ery­thromycin	pDG1664 pB­S2E	25 mg/​ml	wa­ter	12.5 μg/​ml
Chloramphenicol	se­lec­tion of trans­for­mants in B.subtilis	pDG3661 pB­S1C	50 mg/​ml	ethanol	5 μg/​ml

Bacillus sub­tilis TRANSFORMATION #

Day 1

1. Streak out cells on ap­pro­pri­ate se­lec­tive me­dia for sin­gle colonies.
2. Use a sin­gle fresh (no more than 18hr old) colony to in­oc­u­late 1ml of 1X MC trp phe with 3mM MgS04 in a 15ml cul­ture tube (900ul H20, 100ul 10X MC, 3ul 1M MgS04, 4ul trp and 4ul phe)

Place on roller­drum at 37°C for 4 hrs.

At 4 hours pre­pare 5ml cul­ture tubes with DNA.

• ge­nomic DNA add 2ul of work­ing, 1:20 and 1:400 di­lu­tions
• plas­mid DNA add 1-2 ul of work­ing stock for Campbell
• plas­mid 1ul and 19ul split of RE di­gest for dou­ble cross-over

3. At 4.5 hrs add 200ul cells to each tube with DNA. Roll all tubes (including no DNA con­trol) for 2 hrs.(One hour is of­ten suf­fi­cient).At this time pre warm the plates to 37°C.
4. Plate on ap­pro­pri­ate se­lec­tive me­dia.
5. 10X MC buffer prepa­ra­tion.

Component	100 mL	200 mL	500 mL
K2HP04	10.7g	21.4g	53.6g
$K{H}_{2}P{O}_4$	5.2g	10.5g	26.2g
Glucose dex­trose	20g	40g	100g
Na3C6H507*2H20	0.88g	1.8g	4.4g
1OOOX Ferric Ammonium Citrate	1ml	2ml	5ml
Casein Hydrolysate	1g	2g	5g
Potassium Glutamate mono­hy­drate	2.2g	4.4g	11g
dd$H_{2}0$	100ml	200ml	500ml

• Mix every­thing us­ing -half the fi­nal vol­ume of wa­ter.
• Once every­thing is dis­solved, ad­just to the ap­pro­pri­ate fi­nal vol­ume.
• Filter ster­il­ize us­ing screw cap fil­ter and ap­pro­pri­ate sized bot­tle. Dis­trib­ute into 10 ml aliquots (in 15ml con­i­cal tubes) us­ing ster­ile tech­nique.
• Label tubes “10X MC”.
• Store in door of -20·c freezer.

1OOOX Ferric Ammonium Citrate - 100ml

Ferric Ammonium Citrate - 2.2g

ddsu to - 100ml

• Filter ster­il­ize us­ing screw cap fil­ter and 125ml bot­tle.
• Wrap in foil(light-sen­si­tive).

[$K_{2}HP{0}_4$ (potassium phos­phate diba­sic an­hy­drous) FW 174.2]

[$K{H}_{2}P{O}_4$ (potassium phos­phate) monoba­sic an­hy­drous) FW 136.1]

[$N{a}_3{C}_6{H}_5{0}_7$ $2H_{2}0$ (sodium cit­rate di­hy­drate) FW 294.1OJ

NOTE: Do NOT use the potas­sium phos­phate diba­sic tri­hy­drate (${K2HP04}$$3H_20$)

Cell lysate prepa­ra­tion for colony PCR - Bacillus sub­tilis #

Suspend a sin­gle colony or cells from a patch in 20 $\mu L$ of ly­sis mix­ture and sub­ject them to the fol­low­ing pro­to­col in a ther­mal cy­cler.

Lysis mix­ture

10 mM Tris-Cl (pH 8.5) - 19.67 $\mu L$ Lysozyme (100 mg/​mL of dd$H_{2}O$) - 0.2 $\mu L$ Proteinase K (800 U/mL) - 0.13 $\mu L$

Protocol 37 0C - 30 min­utes 75 0C - 15 min­utes 95 0C - 05 min­utes 10 0C - $inf$

Store the cell lysate in $-20\mathrm{°}C$.

Genomic DNA ex­trac­tion #

1. Inoculate 3 ml LB with a fresh (w/in 18 hr old) colony.
2. Grow in roller­drum at 37 C for about 3 hours.Tur­bid but NOT over­grown.Pel­let cells in a 1.5ml mi­crofuge tube.Pour off su­per­natant and add re­main­ing cul­ture.(Cell pel­lets may be stored at -20°C).1000 / min 1 s
3. Resuspend cells in 500ul ly­sis buffer.20mM Tris pH 7.5 50mM EDTA 100mM NaCl
4. Add 50ul of 20mg/ml lysozyme (made fresh!).Flick tube to mix.
5. Incubate at 37°C for 10-15 min. If the cells were har­vested from the sta­tion­ary phase, in­cu­bate longer (up to 30 min).
6. Add 60ul 10% sarko­syl (N-lauroylsarcosine).Vortex to mix.(The sus­pen­sion should be­come clear. If it does not, then the cell wall was not de­graded prop­erly by the lysozyme-do not con­tinue - start over).
7. Add 600ul buffered phe­nol. Vortex vig­or­ously for 10-15 sec. Do not worry about shear­ing the DNA.
8. Spin in a mi­crofuge for 5 min­utes, max rpm.
9. Remove aque­ous phase to a fresh tube.Use a 1ml pipette tip that has been cut -0.5cm from the bot­tom - this helps to pull up the chro­mo­so­mal DNA with­out dis­turb­ing the in­ter­face.
10. Add 600ul phe­nol/​chlo­ro­form. Vortex vig­or­ously 10-15 sec.
11. Spin in a mi­crofuge for 5 min­utes, max rpm.
12. Remove aque­ous phase to a fresh tube.Use a 1ml pipette tip that has been cut -0.5cm from the bot­tom.
13. Add 1/10 vol 3M NaOAC. Vortex to mix
14. Add 2 vol TOH.
15. Invert the tube un­til the DNA pre­cip­i­tates as a fluffy white mass.
16. Spin in mi­crofuge for 1 min.Re­move su­per­natant. Add 150ul 70% EtOH, vor­tex, spin 1 min.
17. Remove su­per­natant.Let the pel­let air-dry for 5-15 min.
18. Do not al­low the pel­let to dry out com­pletely, as it will not be pos­si­ble to re­sus­pend.Re­sus­pend DNA in 200ul H-it can be very dif­fi­cult to get the DNA back into so­lu­tion at this stage.
19. Prepare a work­ing stock by mak­ing a 1:10 di­lu­tion (20ul DNA + 180ul TE).Take a wave­length scan 220-340 am.Store at -20°C.
20. Make 1:20 and 1:400 di­lu­tions for 1 XMC trans­for­ma­tions from the work­ing stock.

Protocols for Bioplastics #

Part 01: Extraction of Vanillin from Wheat Straw us­ing the Nitrobenzene #

Oxidation (NBO) Method

Materials Required

Wheat Straws were col­lected from lo­cal fields (Villages Bakaniya and Barkheda Salem) near IISER Bhopal.Various chem­i­cals used in the ex­per­i­ments in­clude Sodium Hydroxide, Nitrobenzene, Dichloromethane, Vanillin, Sodium Sulfate, Methanol, Hexane, Sulfuric Acid, Hydrochloric Acid, and Distilled Water were ob­tained from Prof.Aasheesh Srivastava’s Lab (Lab No.323), Department of Chemistry, IISER Bhopal.

Procedure

Extracting Lignin out of Wheat Straw

1. 10 grams of washed straw was taken and ground with the grinder to ob­tain $NaOH$ and Sulphidity of 30% Sodium Sulfide by weight in the 3:1 ra­tio of $NaOH$ and $N{a}_{2}S$ was pre­pared..

2. 10 grams of Straw, mixed with white liquor and Straw in a 6:1 ra­tio.As­sum­ing the den­sity of the Water to be 1 g/​ml, 60ml of the white liquor is re­quired.11.25g of $NaOH$ pellets in 45ml of Distilled Water and 4.5g of $N{a}_{2}S$ in 15ml of Distilled Water was taken and added it to the Round Bottom flask con­tain­ing the Straws.

3. Refluxed the above mix­ture at 160°C in an oil bath for 3 hours

4. After re­flux­ing, a dark brown col­ored vis­cous so­lu­tion was ob­tained, called Black liquor, and the residue of un­treated Straw was col­lected at the bot­tom.

5. Filtered the above so­lu­tion us­ing Vacuum fil­tra­tion to re­move the residue, and black liquor is col­lected as the fil­trate.

6. The pH of the Black Liquor was tested us­ing fil­ter pa­per.The pH was found to be around 13.

7. The Black liquor is neu­tral­ized us­ing 98% pure $H_{2}S{O}_4$ (for our ex­per­i­ment, we took 7ml) and fur­ther acid­i­fied un­til the pH of the so­lu­tion reaches 2 — 3 (pH pa­per turned pink).pH 2 was nec­es­sary to in­crease the ef­fi­ciency of Lignin.

8. On neu­tral­iza­tion, the color of the so­lu­tion was changed from Dark Brown to Peanut Brown Colour, and pre­cip­i­ta­tion was ob­served.

9. Solution was left overnight, and the next day, so­lu­tion was fil­tered us­ing vac­uum fil­tra­tion and washed it with cold Water 2 — 3 times to re­move ex­cess $H_{2}S{O}_4$.

10. The solid residue was taken out and dried for around 24 hours in a hot air oven at 65°C

11. After 24 Hours, a hard residue of Lignin was formed, which is woody brown, and then Lignin was ground us­ing a Mortar and pes­tle till fine par­ti­cles were ob­tained.Stored the lignin in the Vial by weigh­ing it.

12. We tested the Lignin qual­i­ta­tively us­ing the Safranine dye and re­ported the re­sults (fig­ure 1(k) in re­sults sec­tion).

Synthesis of Vanillin from Extracted Lignin us­ing the NBO method

1. 1.5 grams of dried Lignin was taken in a round bot­tom flask and 52.5ml of 2M $NaOH$ (4.19 grams $NaOH$ in 52.5ml of Distilled Water) and 3.75ml of Nitrobenzene were added.Ni­troben­zene acts as a mild ox­i­diz­ing agent un­der al­ka­line con­di­tions and sup­ports the side-chain ox­i­da­tion of Lignin.
2. Refluxed the above mix­ture in an oil bath for 3 hours at 160°C till a dark brown- col­ored so­lu­tion was ob­tained.
3. After Refluxing, the con­tents from the Round Bottom flask to the sep­a­rat­ing fun­nel were trans­ferred and two lay­ers of liq­uids were ob­tained.
4. Lower layer was sep­a­rated into a beaker and rest was left in the sep­a­rat­ing fun­nel for fur­ther test­ing.
5. The so­lu­tion in the beaker was di­vided into three parts and fol­low­ing tests were per­formed:
   • In Part 1, ran the TLC against Laboratory–grade Vanillin us­ing 20% EtOAc/Hexane so­lu­tion as elu­ent, stained it with 2,4 DNP, and re­ported the re­sults.
   • In Part 2, layer was acid­i­fied with 1M HCl and then ran the TLC of the pre­cip­i­tate against Laboratory–grade Vanillin us­ing 20% EtOAc/Hexane so­lu­tion as elu­ent, stained it with 2,4 DNP, and re­ported the re­sults.
   • In Part 3, so­lu­tion was dis­solved into cold di­ethyl ether and kept overnight, cov­er­ing it with alu­minum foil to get the pre­cip­i­tate.The next day, ran the TLC of the so­lu­tion against Laboratory–grade Vanillin us­ing 20% EtOAc/Hexane so­lu­tion as elu­ent, stained it with 2,4 DNP, and re­ported the re­sults.
   • From Part 3, di­ethyl ether was re­moved by evap­o­rat­ing it and 20 ml of Methanol and wa­ter mix­ture (1:1 ra­tio) was added to the re­main­ing so­lu­tion.Both the lay­ers were im­mis­ci­ble, and a cloudy so­lu­tion was ob­tained.Ran the TLC of the so­lu­tion against Laboratory–grade Vanillin us­ing 20% EtOAc/Hexane so­lu­tion as elu­ent, stained it with 2,4 DNP, and re­ported the re­sults.
6. up­per layer from the sep­a­rat­ing fun­nel was re­moved in step 4 in the beaker and pH of the so­lu­tion was tested.
7. Separated up­per layer was neu­tral­ized us­ing 98% pure HCl till the pH of the so­lu­tion reached around 4.
8. After neu­tral­iza­tion, so­lu­tion was passed through 90mm fil­ter pa­per us­ing Vacuum fil­tra­tion and fil­trate was col­lected in the Buchner fun­nel.
9. Filtrate was trans­ferred into the Separating Funnel, and as the layer was not sep­a­rated, 20 ml of Dichloromethane (DCM) was added.Both the so­lu­tions were mixed by shak­ing and let­ting the lay­ers sep­a­rate with time.
10. Once the lay­ers were sep­a­rated, lower layer of DCM was ex­tracted in the Round Bottom Flask and more DCM was added into the sep­a­rat­ing fun­nel 2 more times to get the com­pound of our in­ter­est in the or­ganic layer and col­lected it in the Round Bottom Flask.
11. Organic Solvent (DCM) was evap­o­rated us­ing the Rotavapor un­der re­duced pres­sure to get the crude prod­uct.
12. The prod­uct was stored in the 5ml Vial and ran the TLC against Laboratory–grade Vanillin us­ing four dif­fer­ent elu­ents, 10% EtOAc/Hexane so­lu­tion, 20% EtOAc/Hexane so­lu­tion, 30% EtOAc/Hexane so­lu­tion and 40% EtOAc/Hexane so­lu­tion, stained them with 2,4 DNP and re­ported the re­sults.

Part 02: Formation of Acetyl fer­ulic acid from Vanillin and fur­ther poly­mer­iz­ing it us­ing Zinc Acetate #

Materials Required

The ex­per­i­ments’ ap­pa­ra­tus and var­i­ous chem­i­cals, in­clud­ing Vanillin, Pyridine, Acetic Anhydride, Sodium Acetate, Zinc Acetate, and dis­tilled wa­ter, were ob­tained from Prof.Aasheesh Srivastava’s Lab (Lab No.323), Department of Chemistry, IISER Bhopal.

Experimental Procedure

1. 1.00 grams of Vanillin was taken in a 25 ml round bot­tom flask and 1.08 grams of Sodium Acetate, 4.7 ml of Acetic Anhydride were added.
2. Refluxed the above mix­ture in an oil bath at 140°C for 24 hours till the brown-col­ored so­lu­tion was ob­tained.
3. After 24 hours, the so­lu­tion was poured over 50 grams of crushed ice into a 100 ml beaker and stirred for 45 min­utes till a brown sticky sub­stance was ob­tained.
4. After stir­ring, the mix­ture was kept overnight in the freezer, main­tained at 4°C.
5. The next day, wa­ter was de­canted off and the pre­cip­i­tate was dis­solved in di­ethyl– ether.Once dis­solved, di­ethyl–ether was re­moved by evap­o­ra­tion.
6. After evap­o­rat­ing di­ethyl–ether, a layer of acetyl fer­ulic acid is formed, in­di­cated by Beige yel­low col­oration, which is then dried in the oven for 24 hours.
7. After 24 hours, the com­pound was poly­mer­ized us­ing Zinc Acetate in 1 mol% of Acetyl Ferulic Acid.
8. The re­ac­tion mix­ture con­tain­ing Acetyl fer­ulic acid and Zinc Acetate as a cat­a­lyst was taken in round bot­tom flask and heated the re­ac­tion to ap­prox. $200\mathrm{°}C$ in an oil bath for 2 hours.
9. After poly­mer­iza­tion, the Round Bottom flask was vac­u­umed to re­move any mois­ture if pre­sent.

UPSCALING #

1. 7.00 grams of Vanillin was taken in a 100 ml round bot­tom flask and 7.56 grams of Sodium Acetate, 33 ml of Acetic Anhydride were added.
2. Refluxed the above mix­ture in an oil bath at 140°C for 24 hours till the brown-col­ored so­lu­tion was ob­tained.
3. After 24 hours, the so­lu­tion was poured over 150 grams of crushed ice into a 250 ml beaker and stirred for 45 min­utes till a brown sticky sub­stance was ob­tained.
4. After stir­ring, the mix­ture was kept overnight in the freezer, main­tained at 4°C.
5. The next day, wa­ter was de­canted off and the pre­cip­i­tate was dis­solved in di­ethyl– ether.Once dis­solved, di­ethyl–ether was re­moved by evap­o­ra­tion.
6. After evap­o­rat­ing di­ethyl–ether, a layer of acetyl fer­ulic acid is formed, in­di­cated by Beige yel­low col­oration, which is then dried in the oven for 24 hours.
7. After 24 hours, the com­pound was poly­mer­ized us­ing Zinc Acetate in 1 mol% of Acetyl Ferulic Acid.
8. The re­ac­tion mix­ture con­tain­ing Acetyl fer­ulic acid and Zinc Acetate as a cat­a­lyst was taken in round bot­tom flask and heated the re­ac­tion to ap­prox. $200\mathrm{°}C$ in an oil bath for 2 hours.
9. After poly­mer­iza­tion, the Round Bottom flask was vac­u­umed to re­move any mois­ture if pre­sent.

Part 03: Formation of Bioplastic us­ing poly­mer­ized Acetyl fer­ulic acid and test­ing it #

1. Once the poly­mer­ized com­pound was dried, the com­pound was ground to get yel­low color pow­der.
2. The pow­der was mixed with 0.9 ml glyc­erol and 1 gram of delig­ni­fied straws and then heated in the mi­crowave for 45 sec­onds.
3. After heat­ing it in the mi­crowave, we molded the plas­tic in the re­quired shape and dried it in a hot air oven at 45°C for 3 days.

BIODEGRADABILITY TEST #

Procedure: Mesh Bag Method

Following are the steps we fol­lowed to test the biodegrad­abil­ity of the bio­plas­tics:

1. 0.1001 grams of bio­plas­tic that was de­vel­oped in the first trial was taken.
2. The bio­plas­tic was wrapped with 5 cm × 5 cm, 0.1 mm mesh cloth.
3. The soil was taken in a con­tainer and made it suf­fi­ciently damp for nat­ural soil bac­te­ria to grow.
4. The mesh cloth con­tain­ing the bio­plas­tic was buried in the soil and wa­ter was sprayed to keep the con­di­tions damp.
5. Temperature con­di­tions are room tem­per­a­ture ~ 25 — 27°C
6. The bio­plas­tic was weighed in reg­u­lar in­ter­vals of 24 hours and re­ported the degra­da­tion re­sults.

BACTERIAL GROWTH AFTER BIOPLASTIC DECOMPOSITION

AIM

To com­pare mi­cro­bial growth from soil sam­ples be­fore and af­ter the de­com­po­si­tion of bio­plas­tic in it.

Materials Required

• Bioplastic
• Soil sam­ples
• Conical tube of 0.9% NaCl
• Pipettes
• LB Agar Plates
• Centrifuge tubes

Procedure

• Take some soil in two boxes, bury bio­plas­tic in one of them, and leave both for 5 days.
• Add 1g of the soil sam­ple from both boxes and add to two con­i­cal tubes con­tain­ing 10ml of ster­ile 0.9% NaCl.
• Shake the tubes vig­or­ously to sep­a­rate the bac­te­ria from the soil par­ti­cles.
• Transfer 500$\mu L$ of su­per­natant from both tubes to mi­cro­cen­trifuge tubes. These are the 100 sam­ples.
• Vortex the tubes for 30 sec­onds to thor­oughly mix the bac­te­r­ial cells.
• Add 100$\mu L$ from 100 di­lute so­lu­tions to 900$\mu L$ of saline to make a 10-1 di­lute so­lu­tion.
• Similarly, make 10-1 and 10-2 di­lute so­lu­tions.
• Then plate 100, 10-1, and 10-2 di­lute so­lu­tions of both the sam­ples on LB agar- con­tain­ing plates.
• Incubate the plates at 37°C for 24 hours.

Proof of con­cept #

Engineered bac­te­ria-me­di­ated lignin degra­da­tion in wheat straw #

Experimental Procedure

(Bacterial Procedure):

Bacterial sus­pen­sion prepa­ra­tion:

1. All the clones of xy­lanase C(BBa_K4382004), and BsDyP (BBa_K1336003) were in­oc­u­lated in LB medium with ap­pro­pri­ate an­tibi­otic overnight. The sec­ondary cul­ture was in­oc­u­lated from this pri­mary cul­ture and grown till O.D 0.6.
2. 1gm of straw was then added to these in­ocu­lums in a to­tal vol­ume of 20ml and kept at $37\mathrm{°}C$ overnight with con­stant ag­i­ta­tion at 200 rpm
3. Following which the re­main­ing straw and the me­dia were fil­tered for fur­ther as­says.

Qualitative Test for lignin in the cul­ture me­dia:

1. The cul­ture me­dia was fil­tered us­ing 90mm fil­ter pa­per from the con­i­cal flasks. The straws were col­lected as residues for fur­ther analy­sis and the cul­ture me­dia was taken in 2 dif­fer­ent fal­con tubes.
2. Culture me­dia from the two cul­tures in fal­con tubes were taken to 2 dif­fer­ent 1.5ml Eppendorf with proper la­bel­ing.
3. Then the cul­ture me­dia in 4 Eppendorf was dyed us­ing Safranin dye and kept for some time.
4. The colour change was ob­served and the colour change was ob­served.

2,4-DNP Test

1. The bac­te­r­ial cul­ture was cen­trifuged at 4000 rpm for 10 min­utes to set­tle the bac­te­r­ial cells down in the form of pel­lets and the re­main­ing so­lu­tion was trans­ferred to fresh fal­con tubes.
2. To con­firm whether the lignin is de­graded or not, the so­lu­tion cor­re­spond­ing to the con­trol and test were taken into two Eppendorf of 1.5 ml each.
3. Both the Eppendorf was filled with 100 $\mu L$ of 2,4 DNP each and the colour change was ob­served.

Quantitative Test via Chemical treat­ment for the de­tec­tion of lignin on the straws we get as residue from above

1. The straws in alu­minum foil were taken and la­beled as the first for 20ml bac­te­r­ial cul­ture straws and sec­ond for 20ml con­trol, and left it in a hot air oven overnight at $57\mathrm{°}C$.
2. The next day, based on the lignin de­tec­tion test us­ing safranin on cul­ture me­dia, straws cor­re­spond­ing to 20ml con­trol and 20 ml were taken for ex­trac­tion of lignin if any.
3. We pre­pared two sets con­sist­ing of 1.125 grams of NaOH and 0.45 grams of Na2S to pre­pare the white liquor and trans­ferred them to two round bot­tom flasks of 25ml con­tain­ing the straws.
4. Both the flasks were set on Reflux at $160\mathrm{°}C$ for 3 hours and let cool for an­other 3 hours.
5. After cool­ing them for 3 hours we neu­tral­ized the re­ac­tion mix­ture us­ing 98% pure H2SO4 and added fur­ther H2SO4 to de­crease the pH from 13 to 2.
6. Once the neu­tral­ized pre­cip­i­tate is cooled, we fil­tered both through Vacuum fil­tra­tion and col­lected the residue.
7. We dried the residue in the hot air oven at $57\mathrm{°}C$ for 24 hours and tested the pres­ence of lignin in the residues us­ing Safranin dye and re­ported the re­sults.

Dry Lab Protocol #

METHOD THAT WE WILL USE TO STUDY THE DECOMPOSITION RATES

The lit­ter bag method #

1. The air–dried wheat straw will be cut into 3 — 5 cm sec­tions and dried at 65°C for 8 hours.
2. 10 grams of wheat straw will be weighed in 10 cm × 10 cm mesh bags.
3. Then we will pre­pare the soil with our en­gi­neered bac­te­ria in it.
4. Two dif­fer­ent soils will be pre­pared - one as a con­trol area con­tain­ing our en­gi­neered bac­te­ria and nor­mal soil with­out our en­gi­neered bac­te­ria.
5. We will in­sert the mesh bags con­tain­ing the straw into these two dif­fer­ent soils and mea­sure the de­com­po­si­tion rate.

Environmental con­di­tions: Water will be reg­u­larly sprayed to keep the soil moist and the op­ti­mum tem­per­a­ture will be main­tained for the bac­te­r­ial con­sor­tium to grow.

DATA TO BE MEASURED:

Before Performing the ex­per­i­ment:

1. Initial el­e­men­tal analy­sis of the soil to get the soil’s Nitrogen (N), Phosphorus (P), Potassium (K), and other ions’ con­cen­tra­tions.
2. The mois­ture con­tent of the soil.
3. Water Percolation rate of the soil.

While per­form­ing the ex­per­i­ment (at reg­u­lar in­ter­vals):

1. Straw mass at reg­u­lar in­ter­vals.
2. During the ex­per­i­ment, ions con­cen­tra­tion of N, P, K, and other ions.
3. Water Percolation rate

Every week, the weight of the sys­tem (Soil + Mesh Bag) shall be mea­sured. Decreased weight will give the analy­sis of gases that es­caped to the sur­round­ings.

References #

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