iGEM 2024 — Team macquarieuni

**May**

Week 1

**Date: 22.05.2024**
Members: Nick, Sara
Transferred 1µL of PUS250-V3 plasmid on to 50 µL of DH10B E.coli. Heat shocked seconds, recovered for 1
hour at 37° C in 100 µL LB.

Members: Nick, Jenny, Faith
Plated cultures of LB-KM at 37° C overnight.

**Date: 23.05.2024**
Members: Jenny
Measured the concentration of plasmid by nanodrop. The result was : DNA: 220 ng/ µL 260/280 =1.9.

**Date : 23.05.2024**
Members: Nick, Faiza
Plasmid preparation done using kit as usual (NEB Kit Monarch)

Week 2

**Date: 28.05.2024**
Members: Nick, Faiza
Plasmid preparation continued.

**June**

Week 1

**Date: 07.06.2024**
Members: Faith
Repeated digestion similar to 28.05.2024.
Then did PCR and 1% agarose gel 1x TAE buffer
Remade PSU250 plasmid

Week 2

**Date: 11.06.2024**
Members: Faith
Golden Gate Reaction (GGR) from 7.6.2024
G-block (amilcp) = 25 ng/µL
Incubated at room temperature

 **Date: 12.06.2024**
Members: Faith
GGR transferred 5ul onto 50ul DH10B plated on LB-KM at 37C

 **Date: 13.06.2024**
Members: Nick, Carmen, Jenny
Repeated 12/6 but using PaqCI

 **Date: 14.06.2024**
Members: Carmen, Nick, Jenny
Spread plate from 13/6 plated to 20x grid on LB-KM at 37C

 **Date: 15.06.2024**
Members: Carmen
Moved 14/6 20x grid plate to bench

 **Date: 16.06.2024**
Members: Carmen
Colonies #1-6 from previous 20x grid plate plated onto LB-KM liquid cultures overnight at 37C

 **Date: 17.06.2024**
Members: Carmen
Plasmid extracted with NEB kit eluted in 50ul MQW
Result: Yield low but quality fine

**July**

Week 1

**Date: 9.07.2024**
Members: Ainaz, Jack
PmQ3A, PmQ3B Golden Gate Reactions
![](https://static.igem.wiki/teams/5443/notebook-9-07-pmq3a-gg.png)
![](https://static.igem.wiki/teams/5443/notebook-9-07-pmq3b-gg.png)

Incubate for 40mins at 37C
10ul of each GGR transferred into DH10B E.coli (50ul) then heat shocked then recovered for 1hr at 37C,
added 1ml of LB to each.
Then plated 100ul and pallet onto LB-KM (4 plates prepared)

 **Date: 10.07.2024**
Members: Ainaz
PmQ3C, PmQ3D Golden Gate Reactions
![](https://static.igem.wiki/teams/5443/notebook-10-07-pmq3b.png)
![](https://static.igem.wiki/teams/5443/notebook-10-07-pmq3d.png)

5ul of each GGR transferred into DH10B E.coli (50ul) then heat shocked then recovered for 1hr at 37C,
added 1ml of LB to each.
Then plated 100ul of the pallet onto LB-KM, incubated at 37C overnight (2 plates prepared)

**Date: 11.07.24**
Member:Jenny
PmQ3B
pellet plate : ~100 blue colonies and 10 white colonies
diluted plate: 30 blue colonies and 1 white colony

PmQ3C
pellet plate: 4 white colonies

PmQ3D
pellet plate: 3 white colonies

PmQ3A
pellet plate: 33 blue colonies and 2 white colonies
100 ul plate: 15 blue colonies
All colonies patched onto LB-KM, and incubated at 37C.

**Date: 11.07.24**
Member: Jenny
GGR: PmQ3A and PmQ3B

PmQ3A
ECH: Ppu : 3.6 ul
FCS: Ppu: 7.2 ul
COMT\_ara: 4.6 ul
Sam5: 6.8 ul
Sam8: 6.8 ul
pUS250: 2.2 ul
10x ligase buffer: 4 ul
GG enzyme mix: 2ul
MQ: 2.8ul
= 40 ul total

PmQ3B
ECH Ppu: 3.6 ul
FCS Ppu: 7.2 ul
COMT\_6714: 4.6 ul
Sam5: 6.8 ul
Sam8: 6.8 ul
pUS250: 2.2 ul
10x ligase buffer: 4 ul
GG enzyme mix: 2 ul
= 40 ul total

Thermocycler cycle = x30 cycles
Step 1: 37C - 5 mins
Step 2: 16C - 5mins
Step 3: 60C - 5 mins
But if left overnight add 4C hold step after 30 cycles and then before you transform, do the Step 3 and
then use the DNA to transform

**Date: 12.07.24**
Member: Nick
All patches have grown. Some show brown diffusable pigment. Melanin?

**Date: 12.07.24**
Member: Nick and Carmen
10 ul of each GG reaction transferred into 50 ul DH10B cells

 **Date: 12.07.24**
Member: Faiza
Making Agar plates
400 ml LB-Km and 400 ml LB-Km cumate. Agar was autoclaved and put in hot air over overnight.
Bot-1 (KM 50 ug/ml) - (KM stock - 50ug/ml - 50x10^3 ug/ml)
400 ul/400ml
Bot-2 (KM + Cumate 0.1MM) (cumate stock = 0.5M)
KM = 400 ul, cumate = 80 ul

**Date: 12.07.24**
Member: Faiza and Carmen
Golden Gate Reaction

PmQ3C
ECH: Ppu : 3.6 ul
FCS: 7.2 ul
COMT\_ara: 4.6 ul
hpaBC: 8.8 ul
Sam8: 6.8 ul
MQ: 0.8 ul
10x ligase buffer: 4 ul
pUS250: 2.2 ul
GG enzyme mix: 2ul
= 40 ul total

PmQ3D
ECH: Str : 3.6 ul
FCS-Str: 7.2 ul
COMT\_ara: 4.6 ul
Sam5: 6.8 ul
Sam8: 6.8 ul
MQ: 2.8 ul
10x ligase buffer: 4 ul
pUS250: 2.2 ul
GG enzyme mix: 2ul
= 40 ul total

Thermocycler cycle = x30 cycles
Step 1: 37C - 5 mins
Step 2: 16C - 5mins
Step 3: 60C - 5 mins
N.B. If left overnight, add 4C hold step after 30 cycles and then before you transform, do the step 3
and then use the DNA to transform.

 **Date: 13.07.24**
Member: Nick
Plates moved to 4C

Week 2

**Date: 16.07.24**
Member: Sara
12 colonies of rach type patched onto LB-Km at 37C

 **Date: 16.07.24**
Member: Jack and Ainaz
From 12.07.24 3C and 3D
10 ul of GGR from 12.07 transformed into BC21 (50ul) and plated into LB-Km (just the pellets after
recovery)

 **Date: 16.07.24**
Member: Jack and Ainaz
RARE inoculated into 5ml LB and 5ml M9 (minimal medium) + glucose overnight at 37C.
This is a test of metabolites produced which will show up as background in HPLC.

 **Date: 16.07.24**
Member: Nick
A1, A2, B1-B12, D1-D3
Plasmid preps from 1st round of PMQ3 clones
Patches from 11.07 inoculated into 5ml LB-Km and grown 37C overnight

 **Date: 17.07.24**
Member: Ainaz, Faiza, and Carmen
Plasmid extracted as usual and eluted in 30ul EB, concentrations (ng/ul) estimated using nanodrop:
A1: 52.5
A2: 48.9
B1: 51.4
B2: 72.1
B3: 58.7
B4: 34.7
B5: 56.2
B6: 79.6
B7: 61.0
B8: 31.4
B9: 54.7 ; 1.90; 1.52
B10: 37.6
B11: 52.9
B12: 43.5 ; 1.87; 1.53
D1: 56.9
D2: 69.2
D3: 46.4
B9 and B12 flagged A260/230 and will still send for sequencing
extracts placed in the iGEM freezer box

 **Date: 17.07.24**
Member: Nick
Second round clones are renumbered as B13-B24 and A3-A14. All patches grew well.
Some brown color visible on patches A5, A7, A8, A9, A10, A12, and A14. Go to 18.07.24 (Nick and Jenny)

 **Date: 17.07.24**
Member: Nick, Ainaz, Faiza, and Carmen
Plasmid preps from second round clones of PMQ3A and PMQ3B
Patches of clones from p14 and C's from p16 are inoculated into 4ml LB-Km overnight at 37C.
PMQ3A = 3-14; PMQ3B = 13-18; PMQ3C = 1-3
Note: PMQ3B plate has patches 13-24. only first 6 inoculated overnight cultures due to high number of
potentials for sequencing.
Can come back and prep the rest later if need be.

 **Date: 18.07.24**
Member: Nick and Jenny
Plasmids extracted from 17.07.24 cultures using Monarch Kit, same as previously
Nanodrop readings (ng/ul)
3A 3: 112
3A 4: 165.2
3A 5: 157.6
3A 6: 150.6
3A 7: 120.9
3A 8: 90.7
3A 9: 121.2
3A 10: 121.8
3A 11: 227.7
3A 12: 99.5
3A 13: 133.2
3A 14: 122.9

3B 13: 261.2
3B 14: 160.1
3B 15: 176.6
3B 16: 180.2
3B 17: 142.3
3B 18: 550.7 - (A260/A280 = 1.90) (A260/A230 = 2.24)

3C 1: 128.8
3C 2: 520.2 - (A260/A280 = 1.89) (A260/A230 = 2.23)
3C 3: 132.7

 **Date: 18.07.24**
Member: Nick
Plasmids continued
AU plasmids sent to Plasmidsaurus for sequencing Batch 1
![](https://static.igem.wiki/teams/5443/notebook-18-07-sequence-number.png)

Week 3

**Date: 22.07.24**
Member: Carmen and Faith
10x white colonies from PMQ3D + from plate 16.07 patched onto LB-Km plate at 37C overnight
RARE strain inoculated into 5ml LB, overnight at 37C for competent prep tomorrow
10ul MQW added to Sam8 tube and vortexed

Sam8 PCR
Sam8 = 2
MQW = 8
NUC 371 = 1.25
NUC 372 = 1.25
Q5 2xMM 12.5
total = 25
Ext time: 1.7kb product x 30 sec/kb = 51 sec - round up to 55s with 3min final extension
Thermocycling protocol: Carmen - Q5 TD to 62

 **Date: 23.07.24**
Member: Faiza
Remake pMQ3C/Sam8 PCR continued
Sam8 PCR product looks good. PCR mix made up to 50ul with MQ, then purified using macherey nagel kit,
elude in 30ul.
nanodrop ready: purified PCR product then diluted to typical gblock concentration (25 ng/ul) using
sterile MQ.
GG reaction then done, same as previous page, substitute sm8 PCR product for sam8 gblock.

ech-ppu: 3.6 ul
fcs-ppu: 7.2 ul
comt-ara: 4.6 ul
hpaBC: 8.8 ul
tal.sam8: 6.8 ul
MQ: 0.8
10x ligase buffer: 4ul
pus250 vector: 2.2 ul
GG enzyme mix: 2.0 ul
reaction cycled using same method as p16

 **Date: 23.07.24**
Member: Faiza
competent cells of RARE E.coli
CCMB solution made: (100mL)
CaCl2. H20: 102g
MnCl2.4H20: 0.4g
MgCl2.6H20:0.2g
KAC: 0.5g
glycerol: 10mL
R.O. water: 90mL
add salts to 90mL water
add glycerol
filter sterile into 2x falcon

 **Date: 24.07.24**
Member: Jack
Competent cells of RARE E.coli continued
Plate of RARE (cold room) used to get 1 big loopful of cells ->1 mL LB -> vortex -> inoculate into 100mL
LB
initial OD600 = 0.09 @ 12.20, 0.213 @ 1:30pm, 0.32 @ 1:45pm
cells pelleted (5min 4000g), respended in 10ml of CCMB solution, pellet, respend in 5ml, repeat step,
pellet respend in 2ml then made 20x 100ul ….. 80C

Sequencing refer to Results section

**August**

Week 1

**Date: 07/08/2024**
Members: Jack
Primers, fixing mutations in PMQ3 plasmids by PCR
![](https://static.igem.wiki/teams/5443/notebook-07-08-pcr-primer.png)

Plasmid 3B clone 16
JRD1 2uL
JRD2 2uL
template DNA 1 uL (1:100 dilution)
Master mix Q4 (2x) 50uL
MilliQ 45uL

Plasmid 3C clone 2
JRD3 2uL
JRD4 2uL
template DNA 1 uL (1:100 dilution)
Master mix Q5 (2x) 50uL
MilliQ 45uL

Plasmid 3D clone 2
JRD5 2uL
JRD6 2uL
template DNA 1 uL (1:100 dilution)
Master mix Q5 (2x) 50uL
MilliQ 45uL

PCR using Q5 polymerase: anneal at 5C above TM, Extension 20sec per kb
Initial denaturation: 98C 30sec, 30x(Denature 98C 10sec, Anneal 65C 20sec, Extend 72C 4 min) Final extension 72C 2min

**Date: 07/08/2024**
Members: Jack, Faiza
Transforming pMQ3A\_7 into E.coli RARE
1uL of plasmid pMQ3A\_7 transformed into 50uL of RARE competent cells
Control: no plasmid added

Recover 1 hour 37C, plate on LB-Km

**Date: 07/08/2024**
Members: Faiza
making LB Agar (25gm per 1000ml) = 10gm/400ml
Bot 1 - plain LB (10gm LB powder + 6gm agar)
Bot 2 - LB-Km (10gm LB + 6gm Agar + Km)
400uL of KM stock solution after autoclaving

**Date: 08/08/2024**
Members: Ainaz
No colonies in any plates
Gel electrophoresis for samples A,B, C - no bands
Retransform 3A7, positive and negative controls plate (E.coli RARE)

**Date: 08/08/2024**
Members: Faiza, Jenny
Fixing mutations in pMQ3B,C,D by PCR attempt 2
Repeat PCRs from 07.08 but increase amount of template and use touchdown PCR

pMQ3B
JRD1 - 2uL
JRD2 - 2uL
pMQ3B\_16 - 1uL
Q5 - 50uL
MQ - 45uL

pMQ3C
JRD3 - 2uL
JRD4 - 2uL
pMQ3C\_2 - 1uL
Q5 - 50uL
MQ - 45uL

pMQ3D
JRD5 - 2uL
JRD6 - 2uL
pMQ3D\_2 - 1uL
Q5 - 50uL
MQ - 45uL

**Date: 09/08/2024**
Members: Nick
Good growth for positive control (pus250) - plates and cells okay
tiny colonies unlike with pMQ3A\_7 - good but slow

Week 2

**Date: 12/08/2024**
Members: Sara
continue PCR from 08.08
B - band of correct size seen, but many other bands are also present
C - very faint band of correct size, other stronger bands also present - PCR discarded
D - smear, no bands present - PCR discarded
Gel rerun with all of sample B (4x25uL) band at 11kb cut out, put at 4C

Redo of PCR C and D
we used less template DNA and increased annealing temperature
1 in 10 dilutions made of each plasmid template, expected concentrations 7ng/uL (D2) 53ng/uL (possible C2?)

C
JRD3 - 2uL
JRD4 - 2uL
pMQ3C\_2 - 1uL
Q5 - 50uL
MQ - 45uL

C1 - 50uL
C2 - 50uL + 1uL DMSO

D
JRD5 - 2uL
JRD6 - 2uL
pMQ3D\_2 - 1uL
Q5 - 50uL
MQ - 45uL

D1 - 50uL
D2 - 50uL + 1uL DMSO

4 PCR samples in total: 1 without and 1 with DMSO each

**Date: 13/08/2024**
Members: Faiza/Sara
pMQ3A\_7 in E.coli RARE
colonies from 07.08 (which look orange) scrapped into 1mL LB. OD600 measured (1/10): 0.297
suspension OD600 = 2.97
inoculated into 2x5mL LB-Km (+200uL tyrosine in SMA saturated solution) to give initial OD= 0.1
![](https://static.igem.wiki/teams/5443/notebook-13-08.png)

**Date: 13/08/2024**
Members: Nick
Cultures now at saturation (OD ~2 so we knowthat 200uL DMSO per 5mL culture is okay- not too toxic)
New cultures made from spread plate (07.08) as above
#1: OD600 = 0.94 (I-)
#2: OD600 = 0.89 (I+)

1:25pm
I+ = 1.41
I- = 1.16

1:40pm
I+ = 1.01
I- = 0.87

2:20pm
I+ = 1.1
I- = 1.39

3:05pm
I+ = 0.122
I- = 0.124

5:00pm
I+ = 0.145
I- = 0.145

**Date: 13/08/2024**
Members: Nick, Ainaz, Jack and Faith
Gel purification of PCR of plasmid pMQ3B
Gel slice from 12.08 purified using kit
weight of gel = 0.48g
amount of buffer NTI = 0.96ug/mL - 960uL - no DNA

PCR of G-blocks
remake plasmid from G-blocks
PCR
NVC371 - 1uL
NVC372 - 1uL
Q5mm - 25uL
FCS-Ppu - 1uL
MQ - 22uL
good PCR product, purified as usual, elute in 50uL = 80ng/uL
Dilute to 25ng/uL at 30C

**Date: 14/08/2024**
Members: Nick
no vanilla smell in induced culture

Week 3

**Date: 22/08/2024**
Members: Faiza
Reconstructing pMQ3B,C,D from G-blocks
![](https://static.igem.wiki/teams/5443/notebook-22-08-b.png)

![](https://static.igem.wiki/teams/5443/notebook-22-08-c.png)

![](https://static.igem.wiki/teams/5443/notebook-22-08-d.png)
cycled with proper cycling 30\*( 5min37℃, 5min 16℃)

**Date: 23/08/2024**
members:Jenny
Golden gate reaction (B,C,D) transferred (heat shock 42 45) into DH10B compound cells (50ul).
10uL to 30C/72hours

Week 4

**Date: 26/08/2024**
Members: Nick
Good growth of pellet plates in all cases (B,C,D) with ~70-80 colonies and almost all white colonies
plates put at 4C

pMQ3A\_7 (1uL) transformed into 100uL E.coli RARE, recovered in 1mL LB, 100uL plated onto 5x plates of LB-Km
no growth (27.08 and 28.08) bad plasmid, method, cells?

 **Date: 27/08/2024**
Members: Jenny
24 clones of each type (3B #19-43, 3C #4-28, 3D #4-28) picked to LB-Km patches - 37C

 **Date: 28/08/2024**
Members: Jenny, Jack
all cultures grew well. centrifuge 4000g 5 min. extract plasmids with monarch kit, elute in 50uL LB
Patched of new pMQ3B clones #19-43
each patch inoculated into 5mL LB-Km in 50mL Falcons- 37C shaking

 **Date: 29/08/2024**
Members: Nick, Carmen
pMQ3A\_7 not much DNA left in tube.added 10uL TE then used 3mL to transform DH10B and RARE comp. cells (20uL DH10B and 50uL RARE)
(+) control - 1uL pUS250
start resuscitation 11:45am in 900uL LB, static, pelleted and plated onto LB-Km (~100uL)
x4 plates in 37C incubator- DH10B +, DH10B\_A, RARE +, RARE -A

**Date: 29/08/2024**
Members: Faiza
![](https://static.igem.wiki/teams/5443/results-29-08-nanodrop.png)

**September**

Week 1

**Date: 05/09/2024**
Members: Jenny, Faith
inoculated 3C patches in 5mL LB-Km broth - 37C shaking

 **Date: 06/09/2024**
Members: Nick
culture pelleted and frozen at -30C

Week 2

**Date: 16/09/2024**
Members: Faith
Construct A transform into RARE (1 negative and 1 test)
2uL pMQA\_7 = 240ng
recover in 1mL LB, dilute pellets onto LB-Km

pMQ3C clones 11, 15, 20 show brown diffusable pigment in patches (27.08)
cell pellets from these 3 cultures plasmid-prepped elute in 30uL LB
Nanodrop:
3C\_11 : 90ng/uL
3C\_15: 45ng/uL
3C\_20: 88ng/uL
2uL of each plasmid transformed into RARE E.coli cells using LB-Km at 30C
**Date: 16/09/2024**
Members: Faith, Jack
![](https://static.igem.wiki/teams/5443/notebook-16-09.png)

**Date: 19/09/2024**
Members: Faith
retransform pMQ3A, 3x pMQ3C + pus250 as control into both RARE and DH10B

x5 pMQ3A - RARE
x5 pMQ3C11,15,20 - RARE
x5 pus250 - RARE

x5 pMQ3A - DH10B
x5 pMQ3C11,15,20 - DH10B
x5 pus250 - DH10B

each reaction split between 2x LB-Km plates: 1 at 30C and 1 at 37C = 20 plates

Plate labels:
RARE at 30C - black
RARE at 37C - purple
DH10B at 30C - blue
DH10B at 37C - red

2uL of each plasmid transformed into 20uL of each competent cell type. Recovered in 1mL LB for 1 hour. cells pelleted 16000gG for 2 mins, ~100uL of each onto LB-Km plates. 10x plates in 30C incubation, 10x plates in 37C incubator
after pelleting, 800uL supernatant removed with pipette to ensure that 200uL leftover [100uL x 2 plates]

Note: plates with no growth from 16.09 were put back into the 30C incubator just in case there is very slow growth by next week

Week 3

**Date: 23/09/24**
Members: Carmen
Retransformation of pMQ3A 3x
pMQ3C continued

plates from 19/09
DH10B @30C - lawns! pMQ3A and pMQ3C's have brown pigment diffused into agar but 3Cs are darker. could be more of same pigment? 3A colonies are closer to normal cream color, 3Cs colonies are markedly brown (along with agar coloration)
RARE @30C - very little growth, 1-2 colonies per plate at most. No agar coloration, but colony morphology varies (some big/small, one looks black/brown) could be contamination or mutants?

DH10B @37 - more lawns! colonies are small but expected when colonies are crowded. 3A has agar coloration (similar to @30C), 3Cs have more darker coloration and colonies are dark brown. accumulation of something?

RARE @37C - only 1x single colony on 3A and 3C\_20 plates. nothing on rest.

pus250 controls wokreek fine for all 4 sets (purple lots of large healthy colonies)

still no growth on plates from 16/09, thrown out

RARE strain is clearly struggling maybe accumulating too many aldehydes, leading to toxicity?

sticking to DH10B for now due to time constraints

Induction
scrape ⅔ of each DH10B @37 plate into 1mL LB and measure OD600
for each plate resuspension, inoculate 5mL Lb-Tyr-Km at 3x different starting ODS: 0.05 and 0.1
confirm initial OD
incubate overnight at 37C
will induce with cumate tomorrow morning, these cells seem to grow slower with our plasmids (compare growth with pMQ's vs pus250)

X=(measured OD/target OD)
uL cells to add = (volume of media uL/ X)

scraped cells were very resistant to resuspending fully so let the resuspension mix sit for ~1 min so largest chunks could settle and used 100uL of supernatant + 900uL LB for OD measurements.
![](https://static.igem.wiki/teams/5443/notebook-23-09.png)
in 37C shaking incubator at ~3:40pm

**Date: 24/09/24**
Members: Carmen
ODs of overnight from 23/09
1/10 dilutions of cultures in Lb-Tyr for readings, x by 10 for “true” measurements
![](https://static.igem.wiki/teams/5443/notebook-24-09-1.png)

all way overgrown (aiming for 0.5). transferred 500uL of each into 5mL fresh Lb-Tyr-Km for outgrowth before induction
after outgrowth, transferred 2.5mL of each into new 50mL falcons for induction - rest left as uninduced controls.

![](https://static.igem.wiki/teams/5443/notebook-24-09-2.png)
induced with cumate (find concentration = 100uM) at 1:50pm, put into 37C shaking incubator overnight

**Date: 24/09/2**
Members: Carmen, Faiza, Jenny
repeat of retransformation of pMQ3A and pMQ3Cs
repeat of 19/09 to confirm that strains have not been mixed up, except only at 37C, omitting 30C set

R1: RARE + pMQ3A
R2: RARE + pMQ3C\_11
R3: RARE + pMQ3C\_15
R4: RARE + pMQ3C\_20
R5: RARE + pus250

D1: DH10B + pMQ3A
D2: DH10B + pMQ3C\_11
D3: DH10B + pMQ3C\_15
D4: DH10B + pMQ3C\_20
D5: DH10B + pus250

2uL of each plasmid transformed into 20uL of each competent cell type. recovered in Lb 1mL for 1 hour. pelleted cells at 16000G 2min, removed 800uL supernatant so 200uL leftover cells, resuspended and 100uL each plated onto Lb-Km.
10x plates in 37C incubator overnight , only patrf 1x 100uL so colony density should be the same as last time

**Date: 25/09/24**
Members: Carmen
Overnight Induction Results
excessive cumate was fine. don't need to redo
pMQ3A: more typical saturated E.coli culture color, but uninduced look slightly darker/denser?
pMQ3Cs: all are very dark brown, almost black! such an enormous difference compared to 3A. uninduced also looks slightly darker than induced
pus250: no noticeable difference between induced and uninduced(good and expected) both are dusky like color

Chemical analysis prep
falcons shaken to fully distribute, then 1mL of each transferred t sterile 1.5mL tubes and spiun down at max speed (21k G) for 10 minutes.
remaining cultures in falcon tubes out in 4C fridge
after spindown, 700uL of supernatant from each tube very carefully transferred to new sterile 1.5mL tubes, put into orange freezer box and into iGEM freezer drawer

Observations:

Smell
pus250 cultures smell like normal E.coli cultures
pMQ3A seems to smell less acrid than typical cultures
pMQ3Cs definitely seem to smell almost sweeter or at least far less bad than typical cultures
asked Ruby W. for opinion on culture smells without telling what each was. she said pus250 smelled normal, pMQ3A was not as strong as normal, and at least pMQ3C\_15 (induced and uninduced) smelled “a little fruity” note: only gace pus250-I, 3A+1, and 3C\_15 +I

Visual
3C cultures very dark brown but after pelletting, the supernatant is still brown but the cell pellets are similar color to 3A - the color is in the supernantant
supernatant color of 3A is basically same as pus250 (3A slightly darker yellow?)
for all 3Cs, the uninduced supernatant is noticebaky darker than induced
no cloudiness in any of the supernatants, different colors are all still clear
note: original samples induced with 500uM cumte. additional set repeated with 100uM climate- these have sticker on them. not needed unless something happens to first set

cumate working stick calcs: from 24/09
dilution solution 1mL 100%EtOH - 0.5TRIS in 50% v/v EtOH
needs to be diluted in same solution dissolved in
50mM cumate working stock: 900uL dilution solution + 100uL 0.5M cumate stock
induction: 5u: 50mM cumate in 2.5mL culture = final cumate concentration of 100uM

**Date: 26/09/24**
Members: Carmen
supernatant samples for chemical analysis submitted to Veronica
retransformation plates from 24/09 look the same as 19/09, confirming that strains were nit swapped -placed back into 37C incubator until tomorrow

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# Gene Parts

We aim to build a pathway for vanillin synthesis in *E. coli* starting from tyrosine. This involves the enzymes tyrosine ammonia lyase (TAL), coumarate-3-hydroxylase (C3H), caffeic acid O-methyl-transferase (COMT), feruloyl-CoA synthase (FCS), and enoyl-CoA hydratase (ECH). A summary of the different basic and composite Parts developed is given in the table below.

We will make four different constructs (Figure 1 and 2) to test the effectiveness of different microbial and plant genes at different points in the pathway. In all these constructs, the biosynthetic genes are divided into two transcriptional units, both under the control of cumate-inducible promoters (the cumate-responsive regulator CymR and the first promoter are provided by the cloning vector pUS250).

![](https://static.igem.wiki/teams/5443/screenshot-2024-10-02-at-9-12-24-pm.png)

Figure 1. Structure of vanillin biosynthesis gene clusters tested here

![](https://static.igem.wiki/teams/5443/results-constructs.png)

Figure 2. Structure of vanillin biosynthesis plasmids tested here

Table 1 Vanillin biosynthesis parts developed in this project

| Part name | Organism source | Enzyme | Function | Comments | Registry Links | References |
| --- | --- | --- | --- | --- | --- | --- |
| **Basic parts** | | | | | | |
| TAL-Sam8 | Saccharothrix espanaensis DSM 44229 | Tyrosine ammonia lyase (TAL) | Deamination of L-tyrosine to 4-coumaric acid | Based on:  [BBa\_I742142,](https://parts.igem.org/Part%3ABBa_I742142)   [BBa\_K2906011,](https://parts.igem.org/Part%3ABBa_K2906011)   [BBa\_K1197011](https://parts.igem.org/Part%3ABBa_K1197011) | [BBa\_K5443001](https://parts.igem.org/Part%3ABBa_K5443001) |  |
| C3H-Sam5 | Saccharothrix espanaensis DSM 44229 | Coumaric acid 3-hydroxylase (C3H) | Hydroxylates coumaric acid to caffeic acid | Based on: [BBa\_I742105,](https://parts.igem.org/Part%3ABBa_I742105)  [BBa\_K238007,](https://parts.igem.org/Part%3ABBa_K238007)  [BBa\_K2906013](https://parts.igem.org/Part%3ABBa_K2906013) | [BBa\_K5443002](https://parts.igem.org/Part%3ABBa_K5443002) |  |
| C3H-HpaBC | Escherichia coli MG1655 | Hydroxyphenylacetate 3-hydroxylase | Hydroxylates coumaric acid to caffeic acid | Based on: [BBa\_K1124011,](https://parts.igem.org/Part%3ABBa_K1124011) [BBa\_K4912001,](https://parts.igem.org/Part%3ABBa_K4912001) [BBa\_K4912008](https://parts.igem.org/Part%3ABBa_K4912008) | [BBa\_K5443003](https://parts.igem.org/Part%3ABBa_K5443003) |  |
| COMT-ara | Arabidopsis thaliana | Caffeic acid o-methyltransferase (COMT) | Methylates caffeic acid to produce ferulic acid | Based on: [BBa\_K2906015](https://parts.igem.org/Part%3ABBa_K2906015) | [BBa\_K5443009](https://parts.igem.org/Part%3ABBa_K5443009) |  |
| COMT-6714 | Synechocystis PCC6714 | Caffeic acid o-methyltransferase (COMT) | Methylates caffeic acid to produce ferulic acid | New Part | [BBa\_K5443010](https://parts.igem.org/Part%3ABBa_K5443010) | [Kunjapur et al 2019](https://pubmed.ncbi.nlm.nih.gov/27067813/) |
| FCS-Ppu | Pseudomonas putida KT2440 | Feruloyl-CoA synthetase | Adds CoA to ferulic acid | Based on: [BBa\_K1129001](https://parts.igem.org/Part%3ABBa_K1129001) | [BBa\_K5443011](https://parts.igem.org/Part%3ABBa_K5443011) |  |
| FCS-Str | Streptomyces sp. V-1 | Feruloyl-CoA synthetase | Adds CoA to ferulic acid | Based on: [BBa\_K4256006](https://parts.igem.org/Part%3ABBa_K4256006) | [BBa\_K5443012](https://parts.igem.org/Part%3ABBa_K5443012) |  |
| ECH-Ppu | Pseudomonas putida KT2440 | Enoyl-CoA hydratase/aldolase (ECH) | Hydrolyses Feruloyl-CoA to vanillin | Based on: [BBa\_K1129002](https://parts.igem.org/Part%3ABBa_K1129002) | [BBa\_K5443013](https://parts.igem.org/Part%3ABBa_K5443013) |  |
| ECH-Str | Streptomyces sp. V-1 | Enoyl-CoA hydratase/aldolase (ECH) | Hydrolyses Feruloyl-CoA to vanillin | Based on: [BBa\_K4256007](https://parts.igem.org/Part%3ABBa_K4256007) | [BBa\_K5443014](https://parts.igem.org/Part%3ABBa_K5443014) |  |

| Composite Parts – Single genes with RBS added | Organism | Enzyme | Function | Part Number |
| --- | --- | --- | --- | --- |
| RBS1-TAL-Sam8 | Saccharothrix espanaensis DSM 44229 | Tyrosine ammonia lyase (TAL) | Deamination of L-tyrosine to 4-coumaric acid | [BBa\_K5443016](http://parts.igem.org/Part%3ABBa_K5443016) |
| RBS2-C3H-Sam5 | Saccharothrix espanaensis DSM 44229 | Coumaric acid 3-hydroxylase (C3H) | Hydroxylates coumaric acid to caffeic acid | [BBa\_K5443018](http://parts.igem.org/Part%3ABBa_K5443018) |
| RBS2-C3H-HpaB C | Escherichia coli MG1655 | Hydroxyphenylacetate 3-hydroxylase | Hydroxylates coumaric acid to caffeic acid | [BBa\_K5443020](http://parts.igem.org/Part%3ABBa_K5443020) |
| RBS3-COMT-ara | Arabidopsis thaliana | Caffeic acid o-methyltransferase (COMT) | Methylates caffeic acid to produce ferulic acid | [BBa\_K5443022](http://parts.igem.org/Part%3ABBa_K5443022) |
| RBS3-COMT-6714 | Synechocystis sp. PCC6714 | Caffeic acid o-methyltransferase (COMT) | Methylates caffeic acid to produce ferulic acid | [BBa\_K5443023](http://parts.igem.org/Part%3ABBa_K5443023) |
| RBS4-FCS-Ppu | Pseudomonas putida KT2440 | Feruloyl-CoA synthetase | Adds CoA to ferulic acid | [BBa\_K5443024](http://parts.igem.org/Part%3ABBa_K5443024) |
| RBS4-FCS-Str | Streptomyces sp. V-1 | Feruloyl-CoA synthetase | Adds CoA to ferulic acid | [BBa\_K5443025](http://parts.igem.org/Part%3ABBa_K5443025) |
| RBS5-ECH-Ppu | Pseudomonas putida KT2440 | Enoyl-CoA hydratase/aldolase (ECH) | Hydrolyzes Feruloyl-CoA to vanillin | [BBa\_K5443026](http://parts.igem.org/Part%3ABBa_K5443026) |
| RBS5-ECH-Str | Streptomyces sp. V-1 | Enoyl-CoA hydratase/aldolase (ECH) | Hydrolyzes Feruloyl-CoA to vanillin | [BBa\_K5443027](http://parts.igem.org/Part%3ABBa_K5443027) |

| Composite Parts – Whole gene clusters | Gene Cluster Composition | Part Number |
| --- | --- | --- |
| pMQ3A | ECH-Ppu-FCS-Ppu-COMT-Ara-C3H-Sam5-TAL-Sam8 | [BBa\_K5443028](http://parts.igem.org/Part%3ABBa_K5443028) |
| pMQ3B | ECH-Ppu-FCS-Ppu-COMT-6714-C3H-Sam5-TAL-Sam8 | [BBa\_K5443029](http://parts.igem.org/Part%3ABBa_K5443029) |
| pMQ3C | ECH-Ppu-FCS-Ppu-COMT-Ara-C3H-HpaBC-TAL-Sam8 | [BBa\_K5443030](http://parts.igem.org/Part%3ABBa_K5443030) |
| pMQ3D | ECH-Str-FCS-Str-COMT-Ara-C3H-Sam5-TAL-Sam8 | [BBa\_K5443031](http://parts.igem.org/Part%3ABBa_K5443031) |

# Cycle 1

## Design

The starting sequences for most of our Parts were pre-existing Parts (see Table 1), with the exception of COMT-6714, for which the sequence was sourced from GenBank. We further improved these Parts as described in the steps below.

**1. Removal of restriction sites.**

To make the Parts as versatile as possible, we removed all the restriction sites from a set of 45 commonly-used Type II restriction enzymes (see end of file for list) - note that these include the iGEM BioBrick and GoldenGate enzyme sets. This was done by making 1-bp changes that do not lead to changes in the amino acid sequence, i.e. silent mutations. Changes were made to codons with frequencies as similar as possible to the original by consulting codon usage tables

**2. Removal of cryptic promoters.**

promoters were predicted using the Salis Lab website, and any predicted promoters with strength >2000 were removed (this was an arbitrary cutoff). This was done by making 1 bp changes at the most conserved positions in the promoter (-35 and -10 regions), while maintaining the protein sequence.

**3. Addition of RBS.**

The extended version of the BBa\_B0034 strong RBS (26 bp) was added to all parts, then the strength of all the RBS's with their attached genes was checked with the Salis Lab tool, including 200 bp upstream and downstream mRNA. (Note that in the case of the first gene in the cluster, the upstream sequence was shorter (44 bp) since the first RBS is close to the transcription start point). The sequences were iteratively modified by 1 bp changes until the RBS strengths were standardised at 10000 ± 3000. Results are shown in Figure 2.

**4. Golden Gate sites.**

BsaI sites were added to each end of the Part, with six unique overhangs to enable assembly of the five-part gene cluster in the pUS250 cloning vector, which already provides two BsaI sites with ends GCCT, TGGC. The remaining overhangs were chosen by the NEBridge GetSet tool, and the complete set of overhangs, in order, was : GCCT, ATTA, CACC, AAGA, CGTC, TGGC

**5. Final checks.**

All the protein-coding sequences in the constructs were re-checked by BLAST-P analysis to ensure that no mutations had been introduced in the previous steps, and by in-silico restriction analysis to confirm all sites were as expected, before ordering the synthetic DNA Parts.

![](https://static.igem.wiki/teams/5443/engineering-seq.png)

Figure 3 Design of RBS for vanillin biosynthesis genes

## Build

We performed four differnet Golden Gate reactions with the 5 gene parts and the pUS250 vector, then transformed the recombinant DNA into *E. coli* DH10B, and then plated on LB-Km.

## Test

The first round of constructs was tested solely by visual inspection of the transformants, and then sequencing of several representative clones. . Plasmid DNA was extracted from pmQ3A to D which clones was subjected to Nanopore sequencing to verify the presence and correct sequence of the inserted genes. This step was crucial to ensure that the genes have been accurately cloned and are positioned correctly within the plasmid constructs.

## Learn

**Phenotypic characterisation.**
Some transformants, especially pMQ3C, yielded colonies of *E. coli* producing a brown diffusible pigment. We hypothesise that this results from the C3H enzyme acting on tyrosine instead of its intended substrate p-coumaric acid. C3H is known to have a very broad substrate range [1]. This reaction of C3H on tyrosine would yield L-DOPA, followed by spontaneous polymerization into a melanin-like compound (See Figure 4).

While this brown colour represents an unintended side reaction, it confirms successful expression of at least one of our foreign genes in *E. coli*, most likely C3H-HpaBC. This observation also indicates that our cumate regulation system is not performing optimally, as these biosynthesis genes should not be active on LB-Km agar without the presence of cumate.

![](https://static.igem.wiki/teams/5443/engineering-melanin.png)

Figure 4 Melanin Biosynthesis

Additionally, we observed that DH10B (pUS250) and RARE (pUS250) exhibited better growth at 30°C compared to 37°C, though this trend was not observed with the biosynthetic plasmids. Interestingly, the brown pigment seemed darker at 30 °C, but colony size remained small at both temperatures.

**Genotypic characterisation**

Sequencing showed that only one of the clones (pMQ3A\_7) matched the expected design perfectly, while all others had either major deletions, or point mutations or both. To try to salvage some useful plasmids from some of the constructs that only had minor errors, we attempted to use PCR and re-ligation to repair these in the next DBTL cycle.

# Cycle 2

## Design

Primers were designed around the mutations in the biosynthetic genes (Figure 1) ; in this diagram, the red marked region is the mutation we are trying to correct.

![](https://static.igem.wiki/teams/5443/engineering-primer.png)

Figure 5 Strategy for repairing mutations in biosynthetic genes

## Build

PCRs to repair the biosynthetic plasmids were done, and where successful, these were purified, self-ligated and transformed *E. coli* DH10B and then plated on LB-Km.

## Test

The second round of constructs was again tested by visual inspection of the transformants and sequencing of several representative clones (as above).

## Learn

The PCR approach for repairing the plasmids was not successful. In some cases, bands of the expected sizes were seen, but these were weak, and many other bands were present. In other cases, no products of the expected sized were obtained. The restrictions on the primer design in this experiment made it hard to make good primers, which aggravates the problem of having very long PCRs (>10 kb).

# Cycle 3

## Design

We decided to repeat the original GoldenGate reactions, and screen more clones to ensure we could find one despite the presence of deletions and mutations. It seems from our earlier DBTL cycles that using the ‘correct’ cycling conditions for GoldenGate helped to yield correct clones in earlier experiments compared to using the ‘cheat’ method (37C incubation only), so this approach (proper temperature cycling) was done for all further work.

We decided also to test the biochemical activity of some of the correct (or hopefully correct!) constructs via cumate induction in tyrosine-enriched broth, then LC-MS analysis. We also hope to be able to identify the brown diffusible pigment via LC-MS.

## Build

As described for cycle 1, we performed Golden Gate reactions with the 5 biosynthetic gene parts and the pUS250 vector, then transformed *E. coli* DH10B, plating on LB-Km. One difference to the earlier cycle is that some of the parts needed to be reamplified by PCR before cloning because stocks were running low.

## Test

The third round of constructs was again tested by visual inspection of the transformants and sequencing of several representative clones (as above).

## Learn

The MS data from the 3C clone 11 indicates successful production of vanillin at a concentration of 0.2 ppm, as evidenced by the observed peak. However, the peak intensities do not correlate precisely with the quantities present, likely due to variations in detector response. In addition to vanillin, we also detected other intermediate substrates within our pathway, including p-coumaric acid, caffeic acid, ferulic acid, and vanillic acid.

These results demonstrate that the TAL and C3H enzymes are functioning effectively, as indicated by the efficient conversion of tyrosine to caffeic acid. However, the accumulation of caffeic acid suggests that the downstream enzymes COMT, FCS, and ECH may not be operating at optimal efficiency, given the presence of ferulic acid and only some vanillin in the MS data. Moreover, the construct included the HpaBC gene, implying that the C3H enzyme from HpaBC is outperforming the Sam5 C3H enzyme.

**References**

1. Ni J, Tao F, Du H, Xu P. Mimicking a natural pathway for de novo biosynthesis: natural vanillin production from accessible carbon sources. Scientific Reports. 2015 Sep 2;5(1).

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The following parts were used in our experiment:

* TAL-Sam8
* C3H-SAm5
* C3H-HpaBC
* COMT-ara
* COMT-6714
* FCS-Ppu
* FCS-Str
* ECH-Ppu
* ECH-Str

A Golden Gate (GG) reaction was performed to insert these parts into the pUS250 plasmid; this was transformed into construction host DH10B E. coli cells cultured on Luria broth agar with kanamycin (LB-Km). Recombinant bacteria colonies selected via blue-white screening were patch-plated onto additional LB-Km plates. Plasmid DNA from colonies on these patch plates was extracted for Nanopore quantification and gene sequencing.

Successfully cloned plasmid DNA was transformed into the expression host RARE E. coli cells. These transformants were transferred from plates into LB-Km broths with tyrosine and allowed to start growing (ideally to mid-log phase) before adding an inducer. Supernatant samples were analysed with liquid chromatography – mass spectrometry (LC-MS).

Detailed protocols for these experimental procedures can be found below.

Making Media and Solutions

LB Basic Recipe: 10 g tryptone, 5 g yeast extract and 5 g NaCl per litre reverse osmosis water

* Make 2 litres by doubling above recipe. Divide into 4 x 400 ml, 1 x 200 ml in 500 ml bottles.

To make LB broths:

1. Divide up one 400 ml broth further into 8 x 50 ml in glass 250 ml flasks, cover with foil. Sterilise by autoclaving alongside the 200 ml LB aliquot (still in its bottle).
2. Allow to cool. Working aseptically, divide up the 200 ml broth into 40 x 5 ml aliquots in 50 ml Falcon tubes, organise in racks, label as ‘LB’ and the date. Ensure labels are visible also on the 50 ml broth flasks. These can all be stored on bench at room temperature.

To make LB agar:

1. Take the other remaining 3 x 400 ml of LB broth from above, and add agar to each bottle to the recommended amount (15 g / L = 6 g / 400 ml). Autoclave. Transfer from autoclave to hot oven (60°C) while still hot. Allow to cool to 60°C.
2. To two of the bottles, add 400 µl of kanamycin stock solution (50 mg/ml) to give a final conc. of 50 µg/ml Km in the agar. This will make two sleeves of LB-Km plates (~2 x 20 plates). Pour the plates in the biosafety cabinet. Label the side of each stack of plates at the end with a single green coloured stripe – this indicates LB-Km. Put back in sleeve, store at 4°C.
3. To another bottle, add 400 µl of kanamycin stock solution (50 mg/ml) and 400 µL of IPTG stock solution (1 M) to give final concs. of 50 µg/ml Km and 1 mM IPTG in the agar. This will make one sleeve of LB-Km-IPTG plates (~20 plates). Pour the plates in the biosafety cabinet. Label the side of the stack of plates at the end with one green and one orange stripe – this indicates LB-Km-IPTG. Put back in sleeve, store 4°C.

TE: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0. Make in reverse osmosis water. Sterilise by autoclaving.

PBS: 8 g NaCl, 1.44 g Na2HPO4, 0.245 g KH2PO4 and 0.2 g KCl per litre of reverse osmosis water, pH 7.4. Sterilise by autoclaving.

CCMB solution for competent RARE E.coli cells: 102 g CaCl2.H20, 0.4 g MnCl2.4H20, 0.2 g MgCl2.6H20, 0.5 g KAC, 10mL glycerol and 90 mL reverse osmosis water. Add salts to water, then add glycerol and filter sterile into falcon tubes.

 Cloning into Cumate Expression Plasmid pUS250

---

 **PCR of pUS250 DNA Plasmid**

---

1. Retrieve the following PCR reagents from the freezers. Thaw all reagents briefly, mix by flicking or vortexing and then put on ice.

* Phusion HF 2x Master Mix
* Primer NVC375 (50 µM)
* Primer NVC376 (50 µM)
* Template DNA
* Sterile MilliQ water

2. Make a 1/100 dilution of the plasmid template (1 µl DNA in 100 µl MQ) for use as the PCR template; keep this on ice. Return the concentrated stock of plasmid to the freezer.

3. Obtain four sterile PCR tubes. Set up the master mix in one of these tubes, as below.

* 100 µl of 2x Master Mix
* 2 µl of NVC375
* 2 µl of NVC376
* 2 µl of dilute template DNA
* 94 µl of MilliQ water

4. Return all source reagents to freezer. Split the 200 µl master mix into 4 x 50 µl between the PCR tubes, ensuring each tube contains 50 µl. Thermocycle according to the below scheme:

1. Initial Denaturation: 98°C for 30 sec
2. Then 30 cycles of: 98°C for 10 sec, 65°C for 10 sec, 72°C for 30 sec
3. Final extension: 72°C for 5 min
4. Then hold at: 15°C

5. Run out 3 µL of the PCR product from one of the tubes on an agarose gel to check for a product. Compare any bands to marker ladder (use 5 µL of pre-made DNA marker). There should be a single strong band at approximatelt 5 kb.

6. If the PCR is successful, pool the products together into one tube and add DpnI enzyme (1 µl). Incubate at 37°C for 15 min. Column-purify the mixture using the NEB kit.

7. Quantify the purified DNA using Nanodrop.

---

 **Golden Gate Reactions**

---

1. Add the reagents (listed in the table below) and mix by gentle flicking.

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Reagents (PMQ3A) | Amount (μl) |  | Reagents (PMQ3B) | Amount (μl) |
| ECH Ppu | 1.8 | ECH Ppu | 1.8 |
| FCS Ppu | 3.6 | FCS Ppu | 3.6 |
| ComT aru | 2.3 | ComT 6714 | 2.3 |
| SAM5 | 3.4 | SAM5 | 3.4 |
| SAM8 | 3.4 | SAM8 | 3.4 |
| PSU250 | 1.1 | PSU250 | 1.1 |
| 10x Ligase Buffer | 2 | 10x Ligase buffer | 2 |
| MilliQ Water | 1.4 | MQW | 1.4 |
| GRG Enzyme | 1 | GRG enzyme | 1 |
| Total | 20 | Total | 20 |

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Reagents (PMQ3C) | Amount (μl) |  | Reagents (PMQ3D) | Amount (μl) |
| ECH Ppu | 1.8 | ECH str | 1.8 |
| FCS Ppu | 3.6 | FCS str | 3.6 |
| ComT aru | 2.3 | ComT ara | 2.3 |
| hpaCB | 4.4 | SAM5 | 3.4 |
| SAM8 | 3.4 | SAM8 | 3.4 |
| PSU250 | 1.1 | PSU250 | 1.1 |
| 10x Ligase buffer | 2 | 10x Ligase buffer | 2 |
| MQW | 0.4 | MQW | 1.4 |
| GRG enzyme | 1 | GRG enzyme | 1 |
| Total | 20 | Total | 20 |

2. Incubate in a thermocycler according to the following conditions: 37°C for 5 mins, 16°C for 5 mins and 60°C for 5 mins, for x30 cycles. If planning to leave overnight, add a 4°C hold step after 30 cycles. Before using the DNA for transformation, incubate at 60°C for 5 mins.\*

\* An alternative shortcut method involves incubating the mixture at 37°C for 40 mins.

---

 **Transformation**

---

1. Add 5 µl of ligation mixture to 50 µl of competent DH10B cells. Incubate on ice for 10 min, heat shock at 42°C for 45 sec, place back on ice 2 min, add 1 ml of LB broth and put at 37°C for 1 hr.

2. Mix by vortexing, then pipette 100 µl onto LB-Km-cumate agar, spread plate with glass beads. Centrifuge the remaining culture (2 min at 16,000 g) then pour off most of the supernatant. Resuspend the cells by vortexing in the last drop of liquid remaining, then plate that culture onto a second LB-Km plate.

3. Incubate plates at 37°C for 16-24hr. Recombinants should form white colonies.

---

 **Digestion and Ligation**

---

1. Make a digestion mixture.

2. Mix by gentle flicking and incubate the digest at 37°C for 30 min. Stop with heat (80°C for 20 min).

3. Add ligase buffer and Hi-T4 ligase enzyme to the above digest, mix by gentle flicking and incubate at 37°C for 60 min.

 Induction

1. Scrape cells from colonies from the plate into 1 mL LB.
2. Measure and confirm initial OD600.
3. Inoculate in 5 mL Lb-Km 5mL LB-Km (+ 200 µL tyrosine in SMA saturated solution).
4. Incubate overnight at 37°C.
5. Add the inducer (100-500 µM cumate) and incubate.

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{% extends "layout.html" %}
{% block title %}
Results
{% endblock %}
{% block page\_content %}
![Results](https://static.igem.wiki/teams/5443/title-results.png)

## **Engineering Cycle 1**

Four constructs (pMQ3A,B,C,D) were designed using different vanillin biosynthetic gene combinations (Figure #1E see Engineering section for more details).
All of the constructs were made in the broad host range expression vector pUS250. The rationale for choosing this cloning vector was 1. The cumate inducer is cheap, non-toxic, and effective, and 2. The plasmids can potentially be easily moved to other hosts apart from *E.coli* in future to test the system further

The first three genes should be expressed from the cumate-responsive promoter in the vector while the second two genes should be expressed from a second cumate-responsive promoter sequence provided with the C3H gene (Sam5 or HpaBC). The use of an inducible promoter system should minimise the metabolic burden of these plasmids on the *E.coli* cells during the construction and other stages.

![](https://static.igem.wiki/teams/5443/results-constructs.png)

**Figure 1.Structure of vanillin biosynthesis plasmids tested here**

A mix of colonies were obtained on LB-Km plates after transforming the Golden Gate reactions into *E.coli* DH10B; most of these were white, providing preliminary evidence for successful cloning since this vector has a blue/white marker function. The overall efficiency of cloning and transformation was variable, with some reactions (pMQ3A,B) yielding dozens of colonies, and others (pMQ3C,D) only a handful.

![](https://static.igem.wiki/teams/5443/results-f2.png)

**Figure 2. Example of representative transformation plates.**

Blue colonies (putative non-recombinant pUS250 vector) have been boxed.

Several representative clones of each type were patched to new plates, and cultures were regrown in LB-Km broth for plasmid extraction and sequencing. The nanodrop data for the resultant extracted plasmids is shown in Tables 1 and 2 below.

**Table 1. Concentrations of pMQ3 plasmid DNA - expt 1.**

![](https://static.igem.wiki/teams/5443/results-17-07-nanodrop-readings.png)

**Table 2. Concentrations of pMQ3 plasmid DNA – expt 2.**

![](https://static.igem.wiki/teams/5443/results-18-07-nanodrop.png)

The figures below illustrate the comparison between our computational design and the actual genetic constructs made in the wet lab. Sequencing analysis of all the clones revealed that only one clone, pMQ3A Clone 7, was flawless, matching the design exactly. All other clones (pMQ3B, C, and D) exhibited either major deletions or minor point mutations (Figures #3, 4, 5, 6) . A summary of the problems with each plasmid clone are provided in Table 3 below.

**Table 3 Summary of plasmid sequencing results from Engineering Cycle 1**

![](https://static.igem.wiki/teams/5443/results-table3.png)

![](https://static.igem.wiki/teams/5443/results-pmq3a.png)

**Figure 3. Alignment of constructed pMQ3A plasmids with expected sequence**

![](https://static.igem.wiki/teams/5443/results-pmq3b.png)

**Figure 4. Alignment of constructed pMQ3B plasmids with expected sequence**

![](https://static.igem.wiki/teams/5443/results-pmq3c.png)

**Figure 5. Alignment of constructed pMQ3C plasmids with expected sequence**

![](https://static.igem.wiki/teams/5443/results-pmq3d.png)

**Figure 6. Alignment of constructed pMQ3D plasmids with expected sequence**

When recombinants were patched from single colonies to new LB-Km plates, it was noticed that some of the pMQ3A and pMQ3C clones produced a brown diffusible pigment (shown for pMQ3C in Figure 7). This was never seen in controls that lacked vanillin biosynthesis genes i.e. DH10B(pUS250) or RARE(pUS250) (see Figure 9)

Based on these results, clone pMQ3A\_7 (correct sequence) was set aside for later phenotypic testing, and some of the pMQ3B, pMQ3C, and pMQ3D clones that had only single point mutation errors were targeted for repair efforts – in theory this can be done by amplifying the plasmids with mutagenic primers, then self-ligating the products.

![](https://static.igem.wiki/teams/5443/results-patch-bcd.jpg)

**Figure 7. Unexpected pigment production from recombinant *E.coli* clones**

## **Engineering Cycle 2**

To address the mutations seen in plasmid clones pMQ3B, pMQ3C, and pMQ3D, we designed specific primers to target the inserted or deleted bases and performed a PCR and self-ligation to restore the intended sequence. The results of these PCRs are shown in Figure 8.

In the first attempt at this PCR, no bands of the correct sizes were seen. In the second attempt, a faint band of the correct size was seen for pMQ3B but many other bands were also present in this PCR. In the third attempt, no correct-sized sized bands were seen although a strong band of the wrong size was obtained from pMQ3C.

![](https://static.igem.wiki/teams/5443/results-3pcr.png)

**Figure 8. PCR of vanillin plasmids to repair mutations.**

Panel A: First attempt on 7 Aug. (standard protocol) Panel B. Second attempt on 8 Aug. (increased amount of template DNA and used touchdown PCR method) Panel C. Third attempt on 12 Aug. (increased annealing temperature and testing DMSO addition)
M: DNA marker, B: PCRs from pMQ3B, C: PCRs from pMQ3C, D: PCRs from pMQ3D

## **Engineering Cycle 3**

We decided to repeat the original GoldenGate reactions, transformations, and plasmid extractions, hoping to find correct clones of types B, C, D using the correct golden gate protocol (2-step cycling) instead of a short cut method (37C 1 h) and via screening more clones of each type by whole-plasmid sequencing. As a step towards optimising the system, we also tested a different expression host (*E.coli* RARE) that has been engineered to enable accumulation of aldehydes (e.g. vanillin) (1), and tested the effect of temperature on growth of the different recombinant clones (Figure 9).

Again in this cycle of testing, the brown pigmentation was seen from DH10B(pMQ3C) clones - three of these (11, 15, 20) were retained for further testing and sequencing. An unexpected finding was that we obtained only a handful of transformants of *E.coli* RARE containing any of the biosynthetic plasmid constructs despite getting good transformation efficiencies with the pUS250 vector with the same batch of competent cells (Figure 9) – this could indicate that the vanillin genes are specifically toxic to the RARE host. Lower temperatures seemed to favour growth of *E.coli* containing the pUS250 vector but did not have a big effect on the recombinant clones.

![](https://static.igem.wiki/teams/5443/results-all-plates.png)

**Figure 9. Growth of Different Recombinant Clones**

Many more new clones of pMQ3B were picked and patched and then grown up for plasmid extractions. Nanodrop data for these plasmids is shown in Table 4. Note that some clone numbers are missing from the set since these cultures did not grow.

**Table 4. Concentrations of pMQ3B plasmid DNA – clones 14 to 44**

![](https://static.igem.wiki/teams/5443/results-29-08-nanodrop.png)

Cultures of pMQ3A\_7 and pMQ3C\_11,15, 20 transferred into LB-Km broth containing tyrosine (0.5 g/l) and cumate (100 µM) to enable full induction of the biosynthetic enzymes and hopefully produce vanillin! Supernatants of these cultures will be used for LC-MS analysis of metabolites.

After overnight incubation, the 3C cultures are very dark brown, while 3A only slightly brown. Interestingly, the 3A and 3C cultures do smell different to control *E.coli* (pUS250) although no-one who volunteered to smell-test thought it smelled like vanilla.

![](https://static.igem.wiki/teams/5443/results-lcms-final.jpg)

**Figure 10: LC-MS of pMQ3C\_11**

Culture pMQ3C\_11 showed a total yield of 0.2ppm vanillin. As shown in Figure 10, several intermediates have also been detected along with vanillin, indicating that the pathway is working. This supports what was seen in Figure 9, which is preliminary evidence for successful expression of the C3H enzyme, which we believe here is making a melanin-like pigment from tyrosine via L-DOPA as an intermediate.

This also shows that the pathway's first two reactions work well involving the enzymes, TAL that converts tyrosine to p-coumeric acid, and C3H that converts p-coumeric acid to caffeic acid. Since there is a significant accumulation of caffeic acid in this pathway, future work should be done to optimise COMT expression.

![E. coli Pom Image](https://static.igem.wiki/teams/5443/ecoli-pom-removebg-preview.png)

**References**

1. Ni J, Tao F, Du H, Xu P. Mimicking a natural pathway for de novo biosynthesis: natural vanillin production from accessible carbon sources. Scientific Reports. 2015 Sep 2;5(1).

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