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From 2014.igem.org

Contents 1 Week 1 2 Week 2 3 Week 3 4 Week 4 5 Week 5 6 Week 6

Week 1

Notes: Unless stated otherwise, all gels contain 2-log ladder

5/9

• •Obtained BW23115 KanR cells- BW23115 cells that had their native CRISPR-Cas system knocked out by the insertion of a Kanamycin resistance gene
  • -Will also be called BW23115 or BW
  • -Conjugated BW23115 KanR cells with contain F’ notation (ex. BWF’)
• •Obtained ER2738 cells that contain the F’ episome (no changes from NEB sample)
  • -Will also be called ER. Assume that all ER samples contain the F’ episome
  • -Streaked sample onto LB+Tet (20ug/mL) to select for colonies containing F’ episome

5/10

• •Did receive colonies from 5/9 selection

Week 2

5/12

• •Need to conjugate BW23115 KanR cells with the F’ episome
  • -Set up overnight cultures of ER2738 and BW23115 KanR
  • -When mixed, ER2738 will donate it’s F’ episome and BW23115 KanR will receive the F’ episome. F’ episome confers Tetracycline resistance

5/13

• •Started M13 Amplification: Amplify M13 phage using the M13K07 Helper Phage
  • -Let precipitated in NaCl/PEG solution overnight
  • -Possible sources of error

Did not sterilize 2.5M NaCl/20% PEG-8000 solution

Added 4-fold PEG solution

• • • • •Compensated by adding more LB

During precipitation, put sample in -20C for 30 minutes before realizing mistake and moving to it to 4C. Sample partially froze

• •Conjugated BW23115 with F’ episome
  • -Added 1mL BW23115 to 1mL ER2738 overnight culture
  • -Incubated at 37C for 30 minutes, shaking
  • -Plated on LB+Kan(50ug/mL)+Tet(20ug/mL)

To select for BW cells that took the F’ episome (containing Tet resistance)

5/14

• •Finished the M13 Amplification
  • -Visualized product on UV-vis. There was a tall spike at 269nm indicating that DNA was present. Did not test at 320nm.
• •Results of BW23115 Conjugation
  • -Many colonies indicating successful conjugation of F’ episome into BW23115
  • -Set up overnight to make freeze down tomorrow
• •Set up overnight of ER2738 to make chemically competent tomorrow

5/15

• •Made freeze down of BW23115 KanR F’
  • -BW23115 E. coli strain with Kanamycin resistance gene inserted into genome and with F’ episome
• •Made chemically competent ER2738 cells
• •Transformation of Litmus28i (from NEB) into chemically competent ER2738 cells
  • -Added 1ul Litmus28i plasmid to 40ul competent cells
  • -Plated on LB + Amp(100ug/mL)
  • -Purpose: To make M13 phage that package Litmus28i DNA. Need phagemid (Litmus28i) DNA in infectable cells (cells containing F’ episome) to introduce M13K07 Helper Phage and make phage.

5/16

• •Results of 5/15 transformation
  • -No growth for No DNA control
  • -Many colonies for sample

Week 3

5/19

• •M13 Amplification to isolate M13-Litmus28i phage
  • -Cells: ER2738 cells containing Litmus28i phagemid
  • -Helper Phage: M13K07
  • -Not much phage was precipitated
• •Set up overnight culture of ER2738 to infect tomorrow

5/20

• •Infected ER2738 cells with M13-Litmus28i phage
  • -Plated only on Ampicillin(100ug/mL) (should have also plated on kanamycin)
  • -Infected for 4-5 hours-> should have only infected for 30 minutes maximum. This extra time gives the cells that were infected with M13-M13K07 the time to produced M13-M13K07 phage and reinfect

5/21

• •Results from M13-Litmus28i infection of ER2738
  • -Solid lawn of growth for diluted and non-diluted
  • -Also sickly looking growth
• •Set up overnights
  • -ER2738 cells containing Litmus28i for freeze down
  • -BW23115 with F’ episome to make chemically competent cells
  • -ER2738 to redo infection

5/22

• •Tested absorbance of phage produced through M13 amplification on 5/19
  • -Low absorbance of 0.018 at 269nm but no detection at wavelength 320nm
  • -Decided to redo M13 amplification
• •Made chemically competent BW23115 with f-episome
• •Made freeze down of ER2738 containing Litmus28i
• •Set up overnight of ER2738 containing Litmus28i to redo M13 amplification tomorrow

5/23

• •Protocol switch to make phage using phagemid
  • -“M13 Amplification” protocol should only be used to make more M13-M13K07, not to make M13 phage containing a different phagemid
  • -Switched to new protocol (“Use of M13K07 Helper Phage for isolation of single stranded phagemid DNA” by NEB. Made modifications (see our protocols) to isolate phage rather than single-stranded DNA)
  • -Making phage….

DATA TABLE HERE******************************************

• •Made fresh antibiotics
  • -Chloramphenicol (34 ng/mL)

1.44g chloramphenicol into 42mL EtOH

• • -Ampicillin (50 ng/mL)

4g ampicillin into 80mL mili-Q H2O

5/24

• •Isolated phage using new protocol
  • -Resuspended pellet in 200ul TBS and 200ul 30% glycerol
  • -Measured absorbance with UV-vis

concentration (phage/mL) = 6x10^16 x (A269-A320)/ (#of base pairs in the phage genome)

DATA TABLE HERE************************************************

• •Infect ER2738 cells with M13-Litmus28i
  • -Wanted 1:10 phage:cell ratio. Math….

At 1 OD (e.coli), cell/mL = 5x10^8

5x10^7 phage * (1mL/4.72x10^12 phage) = 0.011ul phage

• •Set up overnights
  • -ER2738 for infection with M13-Litmus28i
  • -BW23115 F’ for infection with M13-Litmus28i to test infectivity of conjugated strain

Week 4

5/25

• •Infect ER and BWF’ cells with M13-Litmus28i
  • -Made 5mL culture of ER and BW that was at 1 OD

DATA TABLE HERE********************************************************

• • -Based on calculations from 5/24, we needed to add 0.011 ul phage per 1 mL of cells at 1 OD. This equates to 0.055 ul of phage into 5 mL cells; therefore we made a 1:10 dilution so we could add 0.5ul. Unfortunately, the pipet would not take up 0.5ul so we added 0.8ul of M13-Litmus28i phage
  • -Grew the cells for 20 minutes at 37C
  • -Plated 300ul onto Kanamycin (50ug/mL) and 300ul onto Ampicillin (100ug/mL) for each sample

Incubated overnight at 37C

• •Note: During the production of phage, the phagemid SHOULD be packaged preferentially over the Helper Phagemid but some Helper Phagemid will still be packaged. We plated on Amp to select for cells that were infected with phage containing Phagemid. We plated on Kan to select for cells that were infected with phage containing Helper Phagemid. This allows us to compare the packaging efficiency of Helper Phagemid: Phagemid.

5/26

• •Results from 5/25 infection with M13-Litmus28i

DATA TABLE HERE***********************************

• • Conclusions:

Cells grew on Ampicillin; therefore, Litmus28i phagemid was successfully packaged into M13 phage.

For ER2738 samples, there was significant growth on Ampicillin compared to Kanamycin; therefore, Litmus28i phagemid is packaged preferentially over M13K07 Helper Phagemid

M13-Litmus28i retains its infectivity of cells containing the F’ episome

• • -Because we received lawns, we have to redo the infection and plate less cells so we can calculate the uptake ratio between the phagemid and helper phage based on the number of colonies
• •Started 50 mL overnight of K12 ER2738 and BW23115

5/27

• •Redo the infection done on 5/25
  • -Infectable cells: ER2738 and BW23115

Plated non-infected samples of each (non-diluted) to check for contaminants

• • -Diluted M13-Litmus28i (1) phage by a factor of 10. Added 5.5ul to each sample
  • -Grew samples for 20 minutes at 37C, 250rpm
  • -Plated 100ul of onto an Ampicillin (100ug/mL) plate and onto a Kanamycin (50ug/mL) plate. Incubated overnight at 37C.

Dilutions = 1:10; 1:100; and 1:1000

5/28

• •Results from 5/27
  • -Controls were as expected

No growth for ER2738 non-infected grown on Amp, ER2738 non-infected grown on Kan, or BW23115 non-infected grown on Amp

Growth for BW23115 non-infected grown on Kan (BW23115 has Kan R in genome)

• • -Many colonies were received for all dilutions (1:10, 1:100, and 1:1000) of the following

ER2738 infected and plated on Amp

BW23115 infected and plated on Amp

BW23115 infected and plated on Kan

• • -Many (100s to 1000?) colonies grew on 1:10 and 1:100 dilutions of ER2738. 50-100 colonies grew on the 1:1000 dilution of ER2738

Compare this to the 100-200 colonies that grew from 2/25 infection (which was 300ul non-diluted, infected cells)

Reasons for increased yield

•Added too much phage?

•volume changed between experiment (5mL to 50mL)

•Overnight culture may not have been saturated. If still in log phase, the cells would continue to grow

• •Made 50mL O/N cultures of ER2738 and BW23115 so we can repeat the infection tomorrow and plate further dilutions starting at 1:1000
  • -Carry out infection in 5mL and 50mL to test volume effect?

5/29

• •Measured OD of overnights

DATA TABLE HERE**************************************************

• •Experiment 1: Infect cells using same method as 5/25 (in a 5mL culture)
  • -Started with 1OD cells in 5mL
  • -Added about 0.7ul (inaccuracies in pipet) of 1:10 diluted M13-Litmus28i phage
  • -Incubated (rotating) for 20 minutes
  • -Made 1:1,000 and 1:10,000 dilutions
  • -Plated 100ul on Ampicillin (100ug/mL) plates and on Kanamycin (50ugmL) plates

Included non-infected samples diluted by 1:1000

This negative control can be used for Experiment 2 since the non-infected parent solution is the same

• • -Incubate overnight at 37C
• •Experiment 2: Infect cells using protocol from “Eliminating helper phage from phage display”
  • -Diluted O/Ns to OD of 0.1 in 50 mL culture
  • -Grew samples until of OD of ER2728 = 0.59 and the OD of BW23115 = 0.60

Missed OD of 0.5 mark, but the two samples are close to each other

• • -Chilled samples on ice for 30 minutes
  • -Warmed in incubator for 35 minutes (should have been 30)
  • -Amount of phage. Rather than use 1:1 as mentioned in protocol, we used multiplicity of 1:10 (phage:cell)

Added 3.3ul of 1:10 diluted M13-Litmus28i (1) phage

•(On 5/27 we added 5.5ul of diluted phage to 50mL of cells at OD of 1. Our cells were at OD of .6; therefore, 5.5*.6 = 3.3ul)

• • -Incubated for 30 minutes at 37C, not shaking

We later change this to shaking

• • -Dilutions

1:1,000; 1:5,000; 1:10,000; 1:50,000; 1:100,000; 1:1,000,000

Plated ER2738 and BW23115 on Ampicillin (100ug/mL)

Plated ER2738 on Kanamycin (50ug/mL)

• •Experiment 3: Growth Test (for growth curve)
  • -We were concerned by the low OD of the Overnights from the last few days. Wanted to be sure that 2.0-3.0 was not still in log phase. Cultures looked saturated but the OD seemed low.

DATA TABLE HERE ****************************************

47 (369 minutes elapsed) for BW23115 conjugated (without antibiotics) is most likely an error. It has been removed from the growth plot

GRAPH HERE *****************************************************

• •Other
  • -Made Amp and Kan plates (1 sleeve of each)
  • -Made 50mL O/N of ER2738 and BW23115F’ in case we need further dilutions
  • -Made 5mL O/N of ER2738, BW23115F’, and BW23115 (without F’ episome) to make chemically competent tomorrow

Did not have plate of BW23115 (without F’ episome) so used freeze down. Hoping to get O/N of a picked colony from CRISPR Team tomorrow morning

5/30

• •Made chemically competent cells of…
  • -ER2738
  • -BW23115F’ (conjugated with F’ episome)
  • -BW23115 (not conjugated- without F’ episome)

Culture started from plate

• • -BW23115* not conjugated (without F’ episome)

Culture started from freeze down

• •Results from infections
  • -Negative Controls (cells were not infected; cells were diluted 1:1000)

DATA IMAGE******************************************

Results from Experiment 1 (5/29)

DATA IMAGE******************************************

Results from Experiment 2 (5/29)

DATA IMAGE******************************************

Math

If there are 5.00E+8 cells in 1mL of culture at OD of 1, then in 1mL of culture at OD of 0.59, there are 2.95E+8 cells. In a 50mL culture at OD of 0.59, there are 1.48E+10 cells.

We added 3.3ul (0.0033mL) phage at concentration 4.62E+11 phage/mL which amounts to 1.52E+9 total phage

Assuming that 1 phage infects 1 bacterium, we can assume that 1.52E+9 bacterial have the potential to be infected in the 50mL culture

We plated 100ul of culture at various dilutions. If not diluted, the number of cells that can be potentially infected in 0.1mL equals 1.52E+9/500, or 3.05E+06 cells. We then accounted for the dilutions (for 1:1000 dilution, we divided 3.05E+06 by 1000 to receive 3.05E+03)

The following table contains the number of cells with the potential to be infected assuming a 100% infectivity rate by M13 phage and that 1 cell is infected only once.

DATA TABLE*************************************************

• •Conclusions from infections
  • -Results between and within the three trials are inconsistent. For example, the number of colonies received in experiments 1 and 2 from 5/29 differ greatly. Due to the differences in protocol, variation was expected but not to this extent.
  • -Our dilutions did not yield the expected 10 fold (or 5 fold) decrease in growth that was expected.
  • -Plates from 5/29 could be plated better to reduce dense areas of growth and growth around the rim.
  • -Though the experiment contained many errors we can say that the phagemid (Litmus 28i) is preferentially packaged compared to the helper phage (M13K07) but not to the degree we expected.
  • -Could receive increased occurrences of cells containing M13k07 due to infection, phage production, further infection

Week 5

6/2

• •Tested chemically competent cells through transformation

Are cells contaminated?

Are cells competent?

• •The samples for transformation

DATA TABLE *************************************

6/3

• •Results from 6/2 Transformation

DATA TABLE*******************************************

• •The transformations with DNA from the well (B2 and P2) had lower efficiencies than those with DNA from a mini-prep. Most likely this is due to the differences in DNA concentration (p110+RBS (2) 4/23 was at 254.4ng/ul)
• •Conclusions

None of the competent cells were contaminated

All of the competent cells are in fact, competent

• •Set up O/N of DH5-alpha cells to make competent tomorrow

6/4

• •Isolation of single-stranded phagemid DNA using M13K07
  • -Added ER2738 colony to 50mL LB

Plate was cold. Next time warm plate before pricking

•Best to use freshly grown plate

• • -After 4 hours, OD was at 0.02. Waited 45 minutes and OD was at 0.08. Therefore, we infected at OD 0.08
  • -Had started another culture when we did not think the first was growing. In incubator for about 1 hour. OD was 0.00. We infected anyway because last time it worked.
  • -Let infection proceed for 60 minutes then added 70ul of Kanamycin to be a final concentration of 70ug/mL
• •Primers came in to biobrick M13ori (packaging signal on Litmus28i)
  • -Resuspended primers and diluted 1:10

6/5

• •Isolated single-stranded M13K07 DNA
  • -Final concentration = 5724 ng/ul (calculated from a 1:10 dilution)
  • -For second sample in pair, we resuspended it in TE but did not proceed to DNA extraction teps
  • -For the second culture we started 6/4, we resuspended pellet in TBS and glycerol to preserve the M13 phage. Measured absorbances (before glycerol was added)
  • #1

•269 => 1.690A

•320 => 0.103A

• • #2

•269 => 1.453

•320 => 0.059

• •For our first biobrick, we wanted to isolate the M13 origin, a segment ~500bp that allows for packaging into the M13 phage. We tried to achieve this by biobrick assembly and by Gibson Assembly.
  • -To biobrick M13 ori through biobrick assembly (the old-school way)

PCR on Litmus 28i to amplify/biobrick M13ori

•Used primers Gem003 F & R

•Diluted Litmus 28i DNA 1:10

Digestion of p11+RBS (1) to digest pSB1C3 bb with EcoRI-HF and PstI-HF

Ran samples on gel and gel extracted pieces. We recieved very low yields (out of range for nano drop)

•M13 ori: 4.0 ng/ul

•pSB1C3: 1.8 ng/ul

Digested M13 ori fragment despite poor extraction yield with EcoRI-HF and PstI-HF

•Used 1.5x as much DNA as instructed based on inaccurate concentration

GEL IMAGE********************************************************

Gel extracted red rectangles

Ligation

•10hr @ 16C, 10min @ 65C, 4ever @ 4C

• To biobrick M13 ori through Gibson Assembly (the cool-kids way)

PCR on Litmus 28i

•Used primers Gem002 F & R

•Diluted Litmus 28i DNA 1:10

PCR on pSB1C3 (p11+RBS (1))

•Used primers Gem001 F & R

•Diluted pSB1C3 DNA 1:3

6/6

• •Ran gel of PCR products from (6/5). Products will be used for Gibson Assembly
  • -Recieved bands for pSB1C3 around 2000bp and M13ori around 500bp
  • -No contamination in pSB1C3 PCR negative control
  • -Band in M13ori negative control that is the same size as sample. Contaminated by sample?
  • -Gel of PCR products #1 and #2 from 6/5

GEL IMAGE *****************************************

1. pSB1C3 with promoter+RBS as insert. Amplified with Gem001

2. No DNA control for Gem001

3. M13ori amplified with Gem002 from Litmus28i

4. No DNA control for Gem002

• •Gibson Assembly

DATA TABLE *****************************************************

• • -Diluted pSB1C3 and M13ori PCR products 1:10
  • -Incubated 60min @ 50C
  • -Also used provided pUC16 as positive control
• •Transformation
  • 1. p110+RBS Positive control
  • 2. No DNA Negative control
  • 3. Cas9 from distribution kit so we can have more
  • 4. Thaw and refreeze cells Test competency of comp cells after thawed
  • 5. Not chem comp cells Negative control for the above
  • 6. Ligation Product
  • 7. Gibson product

7.2. Gibson product diluted 1:4

• • 8. Gibson positive control

7.2. Gibson positive control diluted 1:4

• • For the Gibson product and the positive control, we transformed 2ul of product and 2ul of 1:4 diluted product. NEB recomends the first if using their competent cells and the second if using cells from other companies. Our cells are from NEB but we made them competent ourselves so we tried both ways

Plated on Chlor at concentrations of 170, 85, and 33 ug/mL

• •Primers came in
  • Resuspended and made 1:10 dilutions

6/7

• •Results from 6/6 transformation
• 1. Positive control

Lots of growth, ~300-400 on 1:10 dilution

• 2. No DNA negative control

No growth

• 3. Cas9 from distribution kit

7 potential colonies (some are close to edges through) on non-diluted

• 4. Thawed then refroze cells

Looks like (1)

• 5. Not chemically competent cells

No growth

• 6. Ligation product

13 potential colonies (some are close to edge)

• 7. Gibson Assembly Product

170 -> No colonies

85 -> No colonies

33 -> 3 specks

• 7.2. Gibson Assembly product diluted 1:10

170 -> 1 speck

85 -> 3 colonies

33 -> 13 colonies

• 8. Gibson positive control

No colonies

• 8.2. Gibson positive control diluted 1:4

No colonies

• •Made 6mL O/N cultures
  • 4 from (3) cas9 plate

See Constitutive CRISPR notebook for more info on these samples

• • 7 from (6) Ligation product
  • 5 from (7.2 [85]) Diluted Gibson product on 85 ug/mL Chlor
  • 8 from (7.2 [33]) Diluted Gibson product on 33 ug/mL Chlor

Week 6

6/8

• •Check colonies for correct constructs.
  • Mini-prepped all 24 O/Ns

Yielded low concentrations for samples 12, 15, 19, and 22

• • Digested all with EcoRI and PstI (10ul reactions)
  • Ran results on gel

All 4 cas9 samples had the expected bands of 2000 and 5000bp

All 7 ligation products have expected bands of 2000 and ~570bp

3 of 5 Gibson assemblies from 85ug/mL Chlor plate had expected bands of 2000 and ~500bp

3 of 8 Gibson assemblies from 33ug/mL Chlor plate had expected bands of 2000 and ~500bp

GEL IMAGE 1-11*********************************************** GEL IMAGE 12-24*********************************************