iGEM 2020 — Team UZurich

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# Notebook

#### July

| Mo | Tu | We | Th | Fr | Sa | Su |
| --- | --- | --- | --- | --- | --- | --- |
|  |  | [1](#0701) | [2](#0702) | [3](#0703) | 4 | 5 |
| [6](#0706) | [7](#0707) | [8](#0708) | 9 | [10](#0710) | 11 | 12 |
| [13](#0713) | [14](#0714) | [15](#0715) | [16](#0716) | [17](#0717) | 18 | 19 |
| [20](#0720) | [21](#0721) | [22](#0722) | [23](#0723) | [24](#0724) | 25 | 26 |
| [27](#0727) | [28](#0728) | [29](#0729) | [30](#0730) | [31](#0731) |  |  |

#### August

| Mo | Tu | We | Th | Fr | Sa | Su |
| --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  |  | 1 | 2 |
| [3](#0803) | [4](#0804) | [5](#0805) | [6](#0806) | [7](#0807) | 8 | 9 |
| [10](#0810) | [11](#0811) | [12](#0812) | [13](#0813) | [14](#0814) | 15 | 16 |
| [17](#0817) | [18](#0818) | [19](#0819) | [20](#0820) | [21](#0821) | 22 | 23 |
| [24](#0824) | [25](#0825) | [26](#0826) | [27](#0827) | [28](#0828) | 29 | 30 |
| [31](#0831) |  |  |  |  |  |  |

#### September

| Mo | Tu | We | Th | Fr | Sa | Su |
| --- | --- | --- | --- | --- | --- | --- |
|  | [1](#0901) | [2](#0902) | [3](#0903) | [4](#0904) | 5 | 6 |
| [7](#0907) | [8](#0908) | [9](#0909) | [10](#0910) | [11](#0911) | 12 | 13 |
| [14](#0914) | [15](#0915) | [16](#0916) | [17](#0917) | [18](#0918) | 19 | 20 |
| [21](#0921) | [22](#0922) | [23](#0923) | [24](#0924) | [25](#0925) | 26 | 27 |
| [28](#0928) | [29](#0929) | [30](#0930) |  |  |  |  |

#### October

| Mo | Tu | We | Th | Fr | Sa | Su |
| --- | --- | --- | --- | --- | --- | --- |
|  |  |  | [1](#1001) | [2](#1002) | 3 | 4 |
| [5](#1005) | [6](#1006) | [7](#1007) | [8](#1008) | [9](#1009) | 10 | 11 |
| [12](#1012) | [13](#1013) | [14](#1014) | [15](#1015) | [16](#1016) | 17 | 18 |
| [19](#1019) | [20](#1020) | 21 | [22](#1022) | [23](#1023) | 24 | 25 |
| 26 | 27 | 28 | 29 | 30 | 31 |

## Notes

---

#### 06/29

Franka

First Day in Lab!
 Transformation of LI plasmids (p1-p34) into competent E.coli cells.
 Plating Agar medium with corresponding antibiotics (add 400 uL of GENT, SPEC, KANA) to the liquid agar.

![](https://static.igem.org/mediawiki/2020/e/ed/T--UZurich--0629.png)

#### 06/30

Elio, Nic

Colony Picking and inoculating LB.
Discuss with Kyle the Primer design (Esp3I vs Bpil). Some plates (18,21,22,25,31,32) were too confluent to pick a single colony, thus the bacteria from these colonies were onto new plates and incubated overnight. Colonies from these plates need to be picked tomorrow.
Made TAP Media (chlamy)
Solution #1: TAP salts solution
Solution #2: Phosphate solution

#### 07/01

Nic, Jonas

TAP Media preparation was continued and colonies picked:
1l was generated, whereby 50ml of the media was added to 4 X 200ml Erlenmeyers respectively, 2x 250ml in a glass bottle (1l total volume) with 3.75g of agar added respectively as these will be used for solid media. The remaining 300ml were also poured into a 1l glass bottle.
Miniprep and colony picking: Colonies except for (18,21,22,25,31,32) get miniprepped (ref. the amplification protocol) colonies on plates 18,21,22,25,31,32 get picked

#### 07/02

Elio, Michelle

Miniprep & transformation of FLS2 and CORE
Performed miniprep of plasmids 18,21,22,25,31 and 32 according to protocol.
Transformed DH10-Beta cells with plasmids containing CORE and FLS2 according to protocol onto Spec plates.
Measure DNA content of miniprep by NANODROP

#### 07/03

Michelle, Jonas

Repeat transformation of FLS2 and CORE, primers arrived today, lated our chlamy
Checked for DNA concentration of the sent CORE and FLS2 DNA in the tubes: The concentration was rather low (12 and 20 ng/μL), but it should be enough
Repeat the transformation according to the protocol with the following difference:
Step 2: add 2 μL of plasmid and put on ice for 30 minutes
Also bacteria that weren't plated were stored at 4 degrees
Scrap off colonies of CC1690 and inoculated a total of 3 plates, inoculated liquid colony in Erlenmeyer Flask as well (120 RPM)

#### 07/06

Elio, Michelle

Picked the two colonies of FLS2 and transferred them to LB.
PCR: As an exercise, we attempted to do an PCR of the 34 Plasmids from Germany, not to sequence but just to amplify. We first identified all primers which could bind to a certain plasmid using Snapgene, then see PCR protocol.

#### 07/07

Elio, Michelle

Ordered primers
Miniprep of yesterday picked FLS 2 nanodrop them: 152.0 ng/μL (1) and 185.1 ng/ μL (2)
Check our PCR products via gel electrophoresis: The PCR products from 06-07 were analyzed according to protocol but did not show nice bands, probably due to miscalculated solution quantities. Anyway one could see that there has been some DNA amplification, and the purpose was to get to know the technique.
Yeast media according protocol, make 40% glucose stock solution

#### 07/08

Elio

Sequencing intro with Kyle, order CORE (via IDT).
The following plasmids with DNA content significantly above the 200 ng threshold should be diluted 50:50 with sterile water:
4(489), 5(437), 6(447), 7(432), 8(405), 10(377), 11(406), 12(379), 14(522), 16(305), 18(305), 19(429), 21(340), 22(510), 25(382), 28(336), 31(387)
5 microlitres of plasmid, 5 of water
Labelling of container: PL #d and stored with the regular plasmids. The sequencing primers are at a 100 micromolar concentration, but should be diluted to 5 with sterile water.Reference "primer dilutions" table for the volume (100 in total), those will be stored together with the other primers.

![](https://static.igem.org/mediawiki/2020/9/99/T--UZurich--0708.png)

#### 07/10

Jonas, Michelle

Sequencing analysis, Chlamy liquid culture, Chlamy hemocytometer
Sequencing general comments: It seems that there is something systemic going on with the ColE1 Seq F primer. My recommendation would be to resequence two or three of the plasmids with this primer that failed - if we get the same results, we can try to design a new sequencing primer ourselves.
Hemocytometer to count cells:
Make 1:10 dilutions: 1 ml of liquid-grown chlamydomonas for the 100 tube and then make 10-1 - 10-3 dilutions with 900 μl of sterile water and 100 μl of the proceeding dilution
Make sure to pipette up and down so that the cells are mixed well in the tube, before pipetting 100 μl to each of the two middle notches.
Count the cells in the 5 big diagonal squares engraved in the hemacytometer.
If there are cells that touch a border: count eg. only top horizontal and vertical left as 1 and rest boundaries as 0: we counted with the 10-3 dilution: 27 cells and 0 cells.

#### 07/13

Elio, Michelle

Transformation of Kyle's plasmids (EFR, BAK1)
Discuss primers for chlamy with Isabel, order chlamy primers
2 overnight cultures: added 5 μl Spec to each, 25 μl E.coli in each tube with EFR and BAK1 DNA, 10 μl of mixed E.coli + DNA to each of the ON tubes
2 plates with Spec: for colony picking tomorrow, 50 μl of mixed DNA + E.coli per plate

#### 07/14

Michelle, Philip

Miniprep BAK1 and EFR
PCR from miniprep BAK1 and EFR
PCR from FLS2(1), primers arrived today
GEL electrophoresis of the PCR products. We need to do PCR for each receptor 2 times since there are always two forward primers (one with and one without signalpeptide) + 1 reverse primer

![](https://static.igem.org/mediawiki/2020/e/ec/T--UZurich--0714.png)

#### 07/15

Philip, Jonas

Purify the PCR products from 07/14
The vector where we clone PCR products in FLS2 is cut with Esp3l and inserted into 02 p641 Esp3l, EFR is cut with Bpil and inserted into 01 p641 Bpil, Bak1 is cut with Esp3l and inserted into 02 p641 Esp3l
Did a new PCR for the FLS2 with signaling peptide (number 8) that failed to produce anything according to the nanodrop measurements.
Changed the polymerisation phase to a length of 3:30, as FLS2 is 3.5 kb long, so the Polymerase has enough time.

#### 07/16

Jonas, Nic

Transformation of E. Coli with Vectors p641-Esp3I: FLS2-, p641-Esp3I : BAK1+- p641-BPiI :EFR+-
Resequence strange results
PCR Gel of FLS2 with SP: The band in the second column is just the primers, if we had our receptor in the PCR product, the band would be much higher (around 3000 bp). We should have a look at our primers and make sure that they are suitable and reconsider the PCR settings, e.g. decrease the annealing temperature, this should make the primers bind more, with the risk of the DNA strands rejoining.

![](https://static.igem.org/mediawiki/2020/9/9d/T--UZurich--0716.png)

#### 07/17

Nic, Michelle

Colony PCR of vector p641-Esp3I: FLS2-, p641-Esp3I : BAK1+- p641-BPiI :EFR+- (use primers of vector and insert)
Redo p641-Esp3I:FLS2 (1) + SP PCR (check Primers, decrease annealing temp.) NOT SUCCESSFUL
Gel:
top row: 1kb ladder | 100bp ladder | E+(1)| E+(2)| positive control | negative control | F+(1) | F+(2)|
bottom row (COLONY PCR): 1kb ladder | 100bp ladder | F-(1)| F-(2) | B+(1)| B+(2)| B-(1)| B-(2)|E-(1)| E-(2)|
B-(2) and E-(2) are the only ones that show appropriate bands. This means that these colonies can be used for the next step!
The rest should be repeated on the next lab day (Monday 20th july)

#### 07/20

Elio, Michelle

Redo Colony PCR of p641-Esp3I: FLS2- , p641-Esp3I: BAK1+ p641-BPiI:EFR+
Redo PCR of p641-Esp3I:FLS2 (1)+ , and p641-Esp3I:FLS2 (2)+
Created a new program with decreased annealing temp: called PCR X7long50
Diluted the arrived for alpha factor peptide and CORE
DIGLIG of CORE and ALPHA signal peptide

#### 07/21

Michelle, Philip

Miniprep and Diagnostic Restriction Digestio (E+ B+ F-)
Overnight cultures: FLS-, BAK1+, EFR+, miniprep
Sequence EFR+ and BAK1+
Compared gel with theoretical bands from Snapgene simulation. The bands are a little messy, but EFR+ and BAK1+ generally look like they should. FLS2; no idea what happened, the rest proceeds to get sequenced.

![](https://static.igem.org/mediawiki/2020/f/f4/T--UZurich--0721.png)

#### 07/22

Philip, Jonas

CORE & MFα transformation into DH10beta E. coli

#### 07/23

Jonas, Nic

Overnight culture of EFR- and BAK1-
Colony PCR of Ll FLS2-
RESULTS: First two columns of both rows show the 1kb ladder. The first 8 columns show the colony PCR results of FLS2-. The next column was not loaded. The next 6 show the results for the CORE colony PCR. On the bottom row, the other two CORE colony PCRs can be seen. Again, one column was left empty, to make it easier to analyze the gel. The remaining 8 show the results for the MFα Colony PCR.
CORE: use colonies 1,3,5,7 and 8. ( = pick 2, should be enough for future work)
FLS2-: not good, but it was advised to still continue
MFa: did work but not very nice observable, use 100 ladder next time and run longer = better separation of the bands -> check with in silico
Sequencing Analysis of Bak1+ and EFR+: Alignments didn't cover the full receptor. In the EFR+ there's a mismatch that changes the amino acid. We should probably pick a new colony for the EFR(+). The BAK1+ needs to be resequenced with different primers that also bind to regions within the receptor sequence.

![](https://static.igem.org/mediawiki/2020/a/a6/T--UZurich--0723.png)

#### 07/24

Elio, Philip

Colony PCR p01:E-, p:02:B-, pouring YPD
Miniprep of p(02, B-) , p(01, E-) was not possible as the overnight culture didn't work
Miniprep FLS2-, MFa, CORE (total 6 as we have 2 cultures/ part)
Pick new colonies for BAK1- and EFR- and do colony PCR: The gel failed completely, only the ladder was visible. Will have to make a liquid overnight culture of all 8 (4 BAK1, 4 EFR) on Monday and do a DRD on Tuesday

#### 07/27

Elio, Michelle

Ordered and design sequencing primers for FLS2 and CORE
Make overnight culture from each two = 4 (2 BAK1-, 2 EFR-) quadrants for DRD on Tuesday
Make more Yeast media for plates

#### 07/28

Michelle, Philip

Miniprep p01:EFR- P02:BAK1-
DRD of ON BAK1- and EFR-
Sequencing (p01:E+,p02:FLS-,p02:CORE, p02:MFa, p02:BAK1-,p01,E-, p02:BAK1+)

![](https://static.igem.org/mediawiki/2020/0/02/T--UZurich--0728.png)

#### 07/29

Philip, Jonas

Colony PCR of EFR- and BAK- -> overnight cultures of them
 Gentamycin plates

#### 07/30

Jonas, Nic

Replating of 16.7 BAK1- onto 2 new plates
Redo colony PCR for BAK1- and EFR- (either from the ones that were plated on the 29.07 or from the original plate)
EFR-: 16.7 (original) was used (8X), BAK1-: replated colonies from 29.07 were used (2X)
Run Gel of colony PCR BAK1- and EFR- -> SUCCESSFUL
The second column contained BAK1-. Here we see a clear, thick band, meaning that this colony PCR worked successfully! EFR-, although using 8 colonies, only worked once, namely in column nb. 6.

![](https://static.igem.org/mediawiki/2020/7/7f/T--UZurich--0730.png)

#### 07/31

Elio, Nic

Make miniprep of liquid culture BAK1+/-, MFα and EFR- (preparation of DigLig)
Redo EFR+ colony PCR -> Successful
po2::BAK1- : sequencing looks fine
po2::BAK1+ : sequencing looks fine
po2::MFα : sequencing looks fine
po1::EFR- : looks empty -> Use overnight culture from 30.07 (successful colony PCR) and MiniPrep it
po1::EFR+: looks empty
What happend to po1::EFR+/- : it seems as if the primer dimers were inserted. do 16 colony PCRs from EFR + to search for the correct insert. Out of 16 colony PCRs, one worked namely nb. 9

![](https://static.igem.org/mediawiki/2020/9/9c/T--UZurich--0731.png)

#### 08/03

Elio, Michelle

Miniprep EFR+, poured YPD plates
Sequence analysis of FLS2- and CORE : CORE fine, FLS2- not so 24 new colonies picked
Check if Kan works as yeast resistance marker: inoculated yeast on one plate with kan and one without -> put in 28 °C incubator overnight

#### 08/04

Elio, Michelle

Sequencing EFR-+, Bak-, FLS-(1), FLS-(2)
FLS2 PCR amplification of both templates (FLS2(1) and FLS2(2))->GEL Purification-> DIGLIG-> then put DigLig in LI p01 in E.coli -> did not work because someone changed the X7long protocol on the machine! We did it again with the correct protocol.

#### 08/05

Elio, Michelle

PCR of FLS2 failed again-> decision with Kyle to order it via IDT
DIGLIG of LII p(10:C) p(10: B+), P(10:B-)
Transformed DH10b to amplify YFP, BB6, BB9
The yeast KAN restistance marker did not work! we will need to use other LII vector than p(11) -> we use dropout medium p(10) with TRP

#### 08/06

Jonas, Nic

Transformation LII p(10:C), p(10:B-), p(10:B+)
Replate YFP, BB6, BB9, restreak of yeast (03.08), restreak chlamy
Seq.Anlys. 04-08:
BAK1 (in p02)(-) : good coverage, looks good
FLS2.1 (in p02)(-): bad coverage, primer dimer insert
FLS2.2 (in p02)(-): bad coverage, primer dimer insert, isn't the end of the world, since we will order FLS2 anyways
EFR (in p01)(-): bad coverage, primer dimer insert
EFR(in p01)(+): bad coverage, primer dimer insert
reamplify from template, gel purification

#### 08/07

Elio, Michelle

PCR Amplification of EFR +/-
Gel product of amplified EFR +/-, with 1kb ladder:-> did not work (Bands are of 500-700 bp.) -> redo PCR Amplification of EFR +/-
Sigma Aldrich: order Yeast Dropout media components
p(10:C), p(10:B+) plates were without any colonies: Since the transformation only worked with p(10:B-) (stored in the fridge), we Repeat DigLig of LII constructs: p(10:C), p(10:B+)

#### 08/10

POWER SHUTDOWN: Overnight cultures BB6, BB9, YFP, BAK1-

#### 08/11

POWER SHUTDOWN: Miniprep of BB6, BB9, YFP, BAK1-
DRD of BAK1-

#### 08/12

Philip, Jonas

Repeat transformation of p10(BAK+) and p10(CORE)
DRD p10(BAK-): inconclusive
Diglig of amplified EFR+ and EFR- into p01 and B+,B-,E- into p10

#### 08/13

Elio, Michelle

DigLig EFR +/-
Transformation: BAK1+/-, CORE in LII and EFR+/- into LI
Overnight cultures of only p(10:B+) since the only one with colonies

#### 08/14

Elio, Nic

Miniprep p(10:B+) & DRD p(10:B+) -> not successful! But plated it anyway to wait for sequencing
DigLig: p(10:CORE) A&B, p(10:BAK1-), p(10:BAK1+)
P(10:B+,B-,C-) and P(01:E-,E+) out of the incubator-> only p(01:E-) successfull
Checkout the dropout: Yeast plates looks fine. there is a little "leakiness" on the ones containing the dropout, however this is okey. No leakiness would be better, however.

#### 08/17

Elio, Michelle

EFR (-) LI colony picking & overnight cultures (use plates preferably from 13.08)
B+ B- LII colony picking and overnight cultures
PCR amplification with the newly arrived primer pr(38) for obtaining Bak1 ectodomain only
Repeat Diglig of LII Bak+,Bak-,CORE
Make new stock dilutions for LII parts
Transformation p(10:B+), p(10:B-), p(10:C) (8 total) (from Fr. 14.08)
Transformation of our todays DIGLIG p(10:B+), p(10:B-), p(10:C) total 6 (from 17-08)

#### 08/18

Michelle, Philip

DigLig new p(10:B+), p(10:B-), p(10:C) -> Transformation of p(10:B+), p(10:B-), p(10:C)
EFR (- ) LI and B(+) B(-) LII each two overnight cultures from 17-08: miniprep, diagnostic restriction (Also do DRD of the p10 plasmid as a control)
Sequence the p10(B-) and p10(B+)
Couldn't pick colonies from 17.8 with p(10:C), p(10:B-), p(10:B+) cause Plates showed no growth
From Bak1 ectodomain PCR from 17.8.20: GEL purification (BAK1 ectodomain is about 850 bp) -> DIGLIG in p02

#### 08/19

Philip, Jonas

DigLig of LI p(01:E-), p(01:E+)
Transformation of Ll DigLig products p(02:B-), p(02:B+), p(02:B\*)
Plates from 18.08 were completely empty today, no colonies
Redo the transformation of yesterdays DigLig with more than 1 microlitre of plasmid

#### 08/20

Jonas, Nic

Sequencing analysis: GFP restriction sites are wrong! Seq matches G53 not G12, we have the wrong GFP!
DigLig:The plates with the Bak ectodomain transformed bacteria from 19-08 were completely empty, so the DigLig has to be redone
Transform EFR from 19-08

#### 08/21

Elio, Michelle

DigLig p(10:B+), p(10:B-), p(10:C) and transformation
DigLig and transform(p02: cheA) (p02: cheB), colony PCR EFR +- and eBak
DigLig split mcherry into p02
Check plates for p(02:E+/-) colonies
Colony PCR: p(02:EFR +/-), Colony PCR: p(02: eBak)
1000 Ladder | 100 ladder | E- E- E- E - E - E+ E+ E+ E+ -> here all E should be a band at 3000 bp only the last EFR seems somehow oke: we streaked out the last EFR + on a plate with gent, no EFR - was working so no plate of that.
1000 Ladder | 100 ladder | eB\* eB\* eB+ eB+ eB-eB- positive control | negative control -> here all bands should be at 1000 bp what they are jupii! we streaked out the eBak s on plates with gent p(P02: eB \*) (p02: eB-) (p02: eB+)

![](https://static.igem.org/mediawiki/2020/e/e8/T--UZurich--0821.png)

#### 08/24

Elio, Jonas

Restreak our yeast on a new YPD plate as the colonies are very big now
Redo colony PCR from p01:EFR- > gel it: gel looks bad, made a new Diglig
p02:eBak pick colonies and transformed it
Repeat LI DigLig of p(01:E-) and transformation
make negative control plate for p02, make overnight cultures of p(01:E+)
EFR showed no amplification: probably wise to drop this plate and wait for todays DigLig and look if that worked
mCherry shows correct amplification, with mCherry A at the bottom left and mCherry B at the bottom right

![](https://static.igem.org/mediawiki/2020/9/92/T--UZurich--0824.png)

#### 08/25

Jonas, Nic

cPCR p(01:E-), yeast transformation p(10:B+/-, C-), DRD LII p(10:B+/-, C-), Sequencing p(10:B+/-, C-), p(02:mCherry), p(eB-/+/\*)
DRD of LII constructs and eBAK
Sequencing of eBAK, LII constructs and p(02:E+), mCherry
colony PCR of p(01:E-): p(01:E+) = Positive results, p(01:E-) = negative results
Redo DigLig of p(02:E+)

#### 08/26

Elio, Jonas

DRD p(10:B+/-, C-) -> All BAK1 columns look splendid! CORE, however, is just weird. We repeat the DRD for CORE
Dilution series for our yeast strain: Do dilution series on selective and non-selective media. Start at OD 0.2 and do a 10fold dilution 5 times = 1, 1:10, 1:100, 1:1000, 1:10000, 1:100000. This should give us a good feel for how much growth ("baseline") we can expect
Diglig LII E-, FLS2 and eBAK for chlamy and transformation

#### 08/27

Jonas, Nic

cPCR p(01:E-), Yeast Transformation (YT) p(10:B-/+, CORE)
Sequencing Analysis:
p(10:B-): looks good
p(10:CORE): no reads at all(?), p(10:B+): strange results, looks like p(10:B-) instead of p(10:B+) as native signal peptide is missing, error could have arisen while setting up the sequencing reaction or the DigLig
p(02:mCherry): 1-159 looks good, 160-235 looks good
p(02:eB+/-/\*): p(02:eB+/\*) both look good, no reads for p(02:eB-)
Yeast transformation p(10:B-)
cPCR p(01:E-) use plate from 24.08-> 1 successful
DigLig: p(02:FLS2), eBAK1- chlamy, p(10:mCherry//eBAK1+/-/\*) (dummies for - and \*, MFα for +)
Overnight cultures of eBAK1-C and FLS2 (from 26.08 plates)
Successful cPCR p(01:E-)
DRD of p(10:CORE-): The "only Scal" and the negative control (no enzymes) look the same. this means that ScaI does not cut, or at least with a very low efficiency! This then results in wierd patterns on the gel. Further, since the sequencing of this plasmid had no reads, we can assume that something with our assembly went wrong. For example in the 2nd column we have 4 bands, one of. course being the uncut, supercoiled plasmid. This means that a multiple of the same insert has been inserted during the assembly.
-> Redo DigLig p(10:CORE)

#### 08/28

Philip, Michelle

DIGLIG and transformation of p(10:Core)
Transformation of p(02:FLS2-), p(02:eBAK1), p(10:mCherry//eBAK1), p(10:B+)
DRD from all the miniprepped overnight cultures-> not good
Asked Isa about the dropout plates: The yeast dropout seemed to work: there was only little growth on the TRP- plate

#### 08/31

Elio, Nic

Plates: all p10(eB-)::splitMcherry look fine p10(eB° mCheA has few colonies though so will not use these)
p2(FLS2) and p2(eBAK) for chlamy are bad, Just smears and no colonies. Kyle said that either the ESP3l enzyme has died or the Gent plate are bad (Gent could have been added too early) yeast with p10(B-): only 2 colonies have grown on 5 plates and one of them looks like Ecoli. The colonies also aren't red, but they should be. Probably means that the transformation failed
Diglig FLS2 and eBAK for chlamy
Overnight cultures from successful p(01:E-) cPCR from 27.05-> miniprep tuesday + sequencing -> thursday Lll
1 Overnight culture of each eBAK+/-/°::McheA/B
4 Overnight cultures of p10( CORE)
4 Overnight cultures of p10(B+)
2 Overnight cultures of p01(E-)

#### 09/01

Elio, Philip

Miniprep everything
DRD everything
split mcherry for chlamy and MFa with Kozak sequence arrived
Diglig split mcherry for chlamy and MFa with Kozak sequence

![](https://static.igem.org/mediawiki/2020/0/05/T--UZurich--0901.png)

#### 09/02

Philip, Jonas

Sequencing: Sequencing eBAK +/-/\* mCherry, CORE and BAK1+ from yesterdays DRD
Take restreaked FLS2- & chlamy eBAK plates from incubator: There was way too much bacteria on the restreak, have to do the restreak again: FLS2, eBAK, mCherry and MFa
Miniprep EFR-
Check negative plate to see if Gent AB works -> Negative was good, no colonies
DRD p(02:E-): bad, no insert
IDT order for all chlamy stuff arrived -> resuspend and nanodrop

#### 09/03

Jonas

DigLig of EFR-Chlamy, CORE-Chlamy, repeat DigLig for E-, Mfa, mCheA-Chlamy, mCheB-Chlamy, eBAK1R and FLS2
Transformation of DigLig products
Our Yeast plates didn't show any yeast again: Kyle will talk with Laura about her yeast transformation protocol and see how it compares to ours.
Colony PCR: Only p02(Mfa-Kozak), p02(mCherA-Chlamy) and p02(mCherB-Chlamy) showed pickable colonies. So a colony pcr was done for those-> mCherB and the Mfa look fine. 2 ONs were made of each.

![](https://static.igem.org/mediawiki/2020/9/9f/T--UZurich--0903.png)

#### 09/04

Nic, Elio

Repeat Diglig for eEFR, eFLS, eCORE, eBAK f. chlamy, CheA f. chlamy
Yeast transformation with CORE, BAK1+, BAK1-mCherry (p10(CORE:YFP A1/B1 and B2) p10(eBAK+::mcheB), p10(eBAK°::mcheA) p10(BAK+::YFP 2 and 3))
Check transformations from 03.09 (p(01:E-), eBAK1-Chlamy, eFLS2, eCORE-Chlamy, eEFR-Chlamy, mCherA, mCherB, MFa)-> Colony PCR EFR-
Sequencing: Only usable p10(BAK::splitmCherry) are p10(BAK°::MchA) and p10(BAK+::MchB), both p10(BAK+::YFP) are good.
p10(CORE::YFP) very interesting. CORE B1/B2/A1 are pretty good except in the YFP, there is apparently an insertion of an Adenine in all 3 reads at the same position. But the L1 plasmid of YFP sequencing doesn't show this insertion, so this is probably just a sequencing error.

#### 09/07

Elio, Michelle

Made a new Diglig for CORE into p02, rest picked colonies and made overnight cultures
Yeast overnight cultures
PCR of Nanoluc with Primers which include the FLAG tag
Cylce purification of PCR of Nanolug with FLAG tag
This GEL looks really nice! all the bands are there to be cut out and purified according the protocol:after the DNA extraction there was no DNA in the samples, something must have went wrong -> Kyle said we should use more DNA (since te PJP32 was also very weak in bands)
PCR amplification to include the FLAG tag: into both the Registry template of Nanoluc and our codon optimized chlamy version was done according PCR protocol as well. To purify this we needed to take a Kit provided by Kyle from the lab called Omega cycle pure Kit. Ee run a GEL anyway to see if it worked, then we did a DRD like Restriction but with the Enzymes that cut only at the ends to make the correct ends for the assembly and we also cut the plasmid PJP32 with the same enzymes (BamHi ad XhoI)
Digest and ligate separate into pJP32
Diglig of CORE::mcheA and McheB
New Diglig of eCORE into p02 -> p(02: C)
Colony PCR from plates 04-09: eBak, eFLS2, eEFR, cheA, PCR of Nanoluc to include FLAG, and cycle purification and GEL purification of chlamy and Reg: the Baks look good, the mcherryA strange-> need to do it again.
FLS2 and EFR size of the fragments 2100 and 2700 look well but sequence anyway so. the nanolucFLAGregistry is fine but the nanolucFLAG chlamy not good but correct size so proceed anyway! ON (Gent Resistance since LII) from all the good stuff: all B1, B4, no mcherry!

#### 09/08

Elio, Michelle

Sequencing of MfaK, mcherryB1, p02, eBak, eFLS2, eEFR for Chlamy miniprep from 04-09, p02 plasmid, p02eBak1, p022Bak4, eEFR (1, 2, 3, 4), eFLS2 (1, 4)
Plate the new eCORE from yesterday
PCR of Nanoluc to insert FLAG Tag in both registry-luc and our codon optimized version chlamy-luc-> did not work again!
another round: PCR for insertion of FLAG into chlamy (need the primer mix 15 (pr 52 and 51) did not work again now for the third time!
Cycle purification after the pcr (of Nanoluc to insert Flag tag) to clean up the pcr Product: see protocol cycle pure kit.
NOTE: The PJP32 colony picked ON culture did not work since our plate is dried out and according to Kyle all colonies are ded so we need to do a new ON culture: we can do this directly with the original plasmid transformed in bacteria, since we know its corect with the original PJP32.
Microscope the yeast ON cultures: Bak+, Bak- both show nice expressions! CORE shows no expression

#### 09/09

Philip, Jonas

Transformation of PJP32 vector into bacteria and streaked onto Carb plates
Colony PCR of p(02:eCORE): The gel was completely empty.
PCR of NLuc-Chlamy: ng/ul yield is very low (5.5 and 4). -> This low a concentration is useless for the next step, something went wrong with the PCR again. We discussed it with Kyle-> change the PCR protocol, longer 1 minute seperation step is so that the GC-rich nanoLuc comes apart correctly, 30 seconds in the normal protocol might be to short.
Digestion: Kyle suggested we should digest all of our PCR amplifications: also have to digest the pJP32. We used the one from Joao and this didn't work very well so maybe try it with the pJP32 that we minipreped. digest about 1 ug of pJP32.
Gel purification : when we did it yesterday, we didn't change the running buffer. Also, when we nanodroped our gel purified stuff, we got a high peak at 230 nm, which indicates contamination, probably because we didn't cut away enough excess gel.

#### 09/10

[Swiss iGEM Meetup](https://2020.igem.org/Team%3AUZurich/Collaboration)

#### 09/11

[Swiss iGEM Meetup](https://2020.igem.org/Team%3AUZurich/Collaboration)

#### 09/14

Elio

Sequencing results: G51 and G52 are the bad GFP with the wrong overhang sequences. G53 is the good YFP, keep it. eFLS: pretty strange: eFLS 1 and eFLS4 have two deletions which would cause frameshift mutations, which means they would be useless. Make Colony-PCR of eFLS2 again. eEFR 1-4: all but number 4 seem pretty good. eEFR 4 has a SNP G -> A, but this is a silent mutation, which chlamy doesn't like, so don't use this one.
eBAK for chlamy 1 and 2. eBAK f. chl. 1 has a large deletion. eBAK f. chl. 2 has a good reverse primer sequence, but the forward read is wrong, there seems to be contamination as you can see multiple peaks overlap. But it still seems that eBAK f. chl. 2 is good, so use this one. p02(McheB for chlamy) is perfect, empty p02 is good
Make Colony-PCR of eFLS2 again and ON (bands 2, 5, 6 and 8 look fine)
Diglig: p11(eEFR::McheA (not for chlamy), p10(eEFR::YFP), p11(CORE::mcheA(not for chlamy)) p10(CORE::YFP) (as the Yeast colonies transfected with this plasmid didn't glow) P2 (McheA for chlamy) p2(CORE) (from the 3 fragments). We need the p11 plasmid be

![](https://static.igem.org/mediawiki/2020/6/60/T--UZurich--09014.png)

#### 09/15

Elio, Nic

Miniprep eFLS2 (4 overnight cultures)
LBit reg, LBit yeast, LBit chlamy, sBit reg, sBit yeast, sBit chlamy, FRB and FKBP12 arrived, resuspend and diglig into P2
Amplification of BAK-::YFPG for in fusion cloning

#### 09/16

Elio

CPCR of p02(mcheA) all the split lucs and the FRB, FKBP12
In Fusion cloning of BAK1::YFP into pJP32 and Diglig of Registry nanoluc into pJP32
Picked first largebit registry, first largebit yeast and second largebit chlamy and second McherryA for chlamy. Because the ladders were quite bad, we couldn't determine the size of the smallbits, so do again. FRBs and FKBP12 very weird, as for example the first and secon FRB have different size, very weird, repeat tomorrow.

![](https://static.igem.org/mediawiki/2020/9/96/T--UZurich--0916.png)

#### 09/17

Elio

Miniprep All Lbits, CheA for chlamy, P11(CORE::MchA), P11(EFR::MchA), p10(CORE:YFP), p10(eEFR:YFP) Lbit yeast and registry didn't grow for some reason, so pick same colony from yesterday again
Colony PCR for Lbit R, all Sbits, p02(eCORE),FRB and FKBP12
Sequence the 4 FLS2
Make overnight culture of WT and p10(BAK::MchB)
Amplify Nanoluc for chlamy with Flagtag primers
Filter sterilize G418
From left to right: 1000 bp ladder, 2x p02(eCORE), 2x Smallbit chlamy, 2x smallbit registry, 2x smallbit yeast, 2x FRB, 2xFKBP12, 2xLargebit registry, 100bp ladder. I made overnight cultures of the SB registry, SB yeast and FRB. eCORE was again negative, suggesting that something is wrong with the IDT ordering. I would suggest dropping it, which isn't too dramatic, as it is only for chlamy.

![](https://static.igem.org/mediawiki/2020/5/5b/T--UZurich--0917.png)

#### 09/18

Elio

Only did 100 ml instead of 300 ml for the 3 hour growing step as we only need to do 4 transformations for each strain, and 100 ml is enough for 5 transformation. Incubated the p10(eBAK for yeast::mcheB) for 4 hours instead of 3, as it grows slower than the WT in YPD the rest of the protocol remains the same. Just discovered that ammonium sulfate inhibits Geneticin selection, and, of course, YNB contains ammonium sulfate, so could only do transformation with the p10 plasmids.
Now that's a great DRD!. Everything is as it should be! From left to right: 1kb ladder, 2xp10(eEFR::YFP), 2xp11(eEFR::MchA)

![](https://static.igem.org/mediawiki/2020/2/2f/T--UZurich--0918.png)

#### 09/21

Elio

Make colony PCR of Nluc registry in pJP32
Make colony PCR of SBit for chlamy and LBit for yeast.
Make digligs of p10(eBAK1::YFP for yeast). Make one Digilig withe the new BB9, P24, p34 and YFP and one diglig with the BB9, p24 and p34 Mastermix (use 3 ul of that) make sure to only use eBAK for yeast and not the one for chlamy
Transformation of DigLig products
Check if yeast colonies have grown->Yeast colonies have grown
Re-amplify Nanoluc for chlamy w. flag tag with the primer NLuc\_R2-F and NLuc\_R2-R, Use anealing temperature of 53°
Make Overnight culture of p10(eBAK::MCheB) in Trp- medium

#### 09/22

Elio, Nic, Michelle

Sequence: Mcherry A for chlamy, eEFR (1)
Do first step of yeast transformation: inoculate 100 ml of -trp with the ON culture
Finish -Trp plates with geneticin (not gentamicin)
Transform yeast: Transformed 2 p11(eEFR::MChA) and p11(CORE::MChA) each into yeast containing p10(BAK::MchB) and plated it onto the freshly prepared -TRP plates + G418
Re Amplify Nanoluc w. FLAG for chlamy using Phusion polymerase
In Fusion cloning: the protocol recommends 100 ng of vector and 50 ng of insert, so added 5 ul of the cut, gel purified pJP32 and 1.5 ul of the amplified BAK::YFP

#### 09/23

Elio

PCR amplify Nluc registry -> did not work!
Clean up Nluc registry and Nluc chlamy
Prepare YPD
Investigate p10(eEFR::YFP) and p10(CORE:YFP) with epifluorescence

#### 09/24

Elio

Make diglig of eEFR::mchb for chlamy, eBAK1::mchA for chlamy. eEFR::SBIT with full length receptor eEFR::SBIT with truncated receptor, eBAK::LBIT with full length receptor, EBAK::LBIT with truncated receptor, CORE::SBIT with full length receptor, CORE::SBIT with truncated receptor
Make dilutions of all the plasmids needed for transformation
ON untransformed yeast in YPD, p10 transformed yeast in -.TRP +geneticin to test effectivity

#### 09/25

Elio

Diglig of yesterdays DNA mixture
Transform yeast with p10(eBAK for yeast:YFP)
PCR of Nanoluc registry to include Flag tag and and reamplification of Nanoluc for chlamy with flagtag with iGEM\_Nluc\_R2- F and iGEM\_Nluc\_R2- R -> The DNA content measured via nanodrop for the reamplified Nanoluc was 10 ng/ul, while the one for the registry nanoluc was 33 ng/ul. Only kept the latter as the former is not enough for successfull cloning.

#### 09/28

Elio

Check the yeast plates of p10(eBAK::YFP)-> looks good
Make colony PCR of the p10 and p11 colonies-> ON
Prepare liquid -TRP medium without Ammonium sulfate YNB Inoculate 2x30 ml of this freshly prepared medium with the co-transformed yeast (one for eEFR, one for CORE)
Re-Amplify Nanoluc for chlamy with FLAG
Transform yeast with the purified plasmids

![](https://static.igem.org/mediawiki/2020/b/b4/T--UZurich--0928-1.png)

![](https://static.igem.org/mediawiki/2020/d/da/T--UZurich--0928-2.png)

#### 09/29

Nic, Michelle, Elio

Autoclave some flasks for ON and transformation of Yeast
DigLigs of p10(eBAK::mCheA)-Chlamy, p11(CORE::SBit)Full, p11(CORE::SBit)Trun
Miniprep the ONCs from yesterday-> Sequence
Purify the nanoluc pcr from yesterday
Yeast ONs: look in Tecan for fluorescene
Transform yeast with the purified plasmids

#### 09/30

Elio

Clean up PCR of Nluc for chlamy from yesterday ->phusion with the EZNA cycle pure kit and got DNA concentration of around 35 ng/ul for both.-> Unfortunately, Nanoluc for chlamy appears as a smear, which makes it unusable. Nluc registry and pJP32 look fine.
Clone nanolucs into pJP32
Begin the western blot for yeast with p10(BAK::MchB)+p11(CORE/EFR::MchA)-> Also prepared 2 SDS-PAGE gel with 15 wells according to the protocol

#### 10/01

Elio

Repeat PCR of Nluc for chlamy with GC buffer
Overnight culture of p10(eBAK::LBit) full 1 and 2, p10(eBAK::LBit) trunc 2 in -Trp
Run SDS gel and transfer it onto membrane
Make overnight culture of pJP32
DigLig: p10(FRB:Sbit) reg and yeast, p8(FKBP12:LBIT), p10(eBAK::YFP) for chlamy
Restreaked p10(eBak::mchB)
As the PCR of Nanoluc for chlamy proved difficult, I decided that I do the digligs for the Split nanoluc codon optimisation experiments. I also made a diglig for p10(eBAK::YFP) for chlamy so that we might make a localisation experiment in chlamy with eBAK

#### 10/02

Elio

Started the transformation of Bak1::Lbit with full length ADH1 promotor yeast with eEFR::Sbit, but was terminated because of western blot result
Treating of Western blot membrane with αFLAG and αHA
PCR of Nanolucs, terminated because it was realised that there aren't any BPs upstreams of the recognition

![](https://static.igem.org/mediawiki/2020/5/5e/T--UZurich--1002.png)

![](https://static.igem.org/mediawiki/2020/1/1a/T--UZurich--1002-2.png)

#### 10/05

Jonas

cPCR of: (SBit:FRB)reg, (LBit:FKBP)reg, (SBit:FRB)yeast, (LBit:FKBP)yeast, (eBAK:mCheA)chlamy, (eBAK:YFP)chlamy
(SBit:FRB)reg -> all fine, pick 1 and 2
(LBit:FKBP)reg -> all fine , pick 1 and 2
(Sbit:FRB)yeast -> all fine, pick 1 and 2
(LBit:FKBP)yeast -> all fine, pick 1 and 2
(eBAK:mCheA)chlamy -> 3 and 4 (maybe 2) are good, pick 3 and 4
(eBAK:YFP)chlamy -> all are good, pick 1 and 2
(CORE:sBit)full -> all have light bands, will pick 1 and 4

![](https://static.igem.org/mediawiki/2020/1/13/T--UZurich--1005.png)

#### 10/06

Elio

Miniprep overnight cultures from yesterday -> sequencing

#### 10/07

Elio

Western blot of eBAK::YFP, BAK::YFP, eBAK::Lbit, eBAK::mchA, eBAK::mchB, (eEFR+eBAK)
Cut pJP32 for inFusion cloning

![](https://static.igem.org/mediawiki/2020/c/cb/T--UZurich--1007.png)

#### 10/08

Elio, Philip

Yeast transformation: p10(eBAK::Lbit) and p10(eBAK::mchA)
Colony PCR of eEFR::YFP in pJP32
Make Carbenicillin LB plates
Confocal imaging: it seems that the whole YFP expression that we saw is just autofluorescence. Since YFP is PH sensitive and it looks like it's in the vakuole we have a problem!

#### 10/09

Elio, Philip

Yeast transformation
colony PCR of eEFR::YFP in pJP32
PCR of eBAK::YFP and eBAK::mchA for in Fusion cloning

#### 10/12

Jonas

Transform bacteria with pJP32
A lot of overnight cultures

#### 10/13

Elio, Philip

Digestion of pJP32 for in-Fusion cloning in chlamy, something went wrong
Dimerization Assay: Compared fluorescent output of Registry split nanoluc and our codon-optimized version of split-nanoluc.

#### 10/14

Elio, Philip

Restriction Digestion of pJP32
Gel purification of the digestion product , cut out the band at 4900 bp -> In-Fusion cloning
Split-NanoLuc measurement: Results showed higher luminescence for the registry split-NanoLuc than for the codon optimized one
Plate reader fluorescence assay of 5 yeast strains transformed with receptors fused to YFP: BAK1+, BAK1-, ectodomain BAK1, ectodomain EFR, CORE. All transformed strains show higher fluorescence than the negative control!

![](https://static.igem.org/mediawiki/2020/a/a7/T--UZurich--1014.png)

#### 10/15

Elio, Philip

Chlamydomonas NanoLuc colony PCR
Western blots of YFP constructs

#### 10/16

Elio, Philip

Dimerization assay of yeast strains transformed with split NanoLuc constructs. There seems to be some increase in chemoluminescence after addition of the appriproate elicitor! The split NanoLuc system might be working!
Confocal miscroscope imaging of the YFP constructs: Membrane localization for EFR and BAK1!

![](https://static.igem.org/mediawiki/2020/0/0b/T--UZurich--1016-1.jpeg)

![](https://static.igem.org/mediawiki/2020/b/ba/T--UZurich--1016-2.jpeg)

#### 10/19

Elio, Jonas

Dimerization assays with EFR & eBAK1 fused to split NanoBit and CORE & eBAK1 fused to split NanoBit

#### 10/20

Elio

Dimerization assay of EFR mcherry and EFR NanoBiT & NanoBiT codon opt.

#### 10/22

Elio

Inoculate -trp medium wit BAK+::YFP, BAK-::YFP,eBAK::YFP,CORE::YFP, eEFR::YFP for imaging tomorrow
FACS measurements

![](https://static.igem.org/mediawiki/2020/d/d6/T--UZurich--1022-1.png)

![](https://static.igem.org/mediawiki/2020/b/b5/T--UZurich--1022-2.png)

#### 10/23

Elio

Confocal imaging of the cultures from yesterday
Centrifuge all tubes at 2000 g for 5 min, remove supernatant, resuspend in 10ml TE buffer. take 1ml into eppendorf, centrifuge at max speed for 10 seconds. take out 950 ml, then add 1 ul of 2.5 mM FM4-64. incubate on ICE for 1 min, then add 1ml TE, resuspend and the centrifuge. Repeat this wash a second time, then resuspend in 50 ul TE. Keep on Ice until microscopy

![](https://static.igem.org/mediawiki/2020/8/8f/T--UZurich--1023-3.png)

![](https://static.igem.org/mediawiki/2020/8/8d/T--UZurich--1023-2.png)

![](https://static.igem.org/mediawiki/2020/1/13/T--UZurich--1023-1.png)

![](https://static.igem.org/mediawiki/2020/2/2c/T--UZurich--logo.png)

### UZurich 2020

[Home](https://2020.igem.org/Team%3AUZurich)
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[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:UZurich/Engineering

iGEM UZurich 2020

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### [iGEM UZurich 2020](https://2020.igem.org/Team%3AUZurich)

# Engineering Success

During our iGEM summer, our team successfully assembled many different composite parts to achieve our goal of using PRRs in a biosensing system. The main goal of these parts was to introduce the PRRs into our host organism *S. cerevisiae* and be able to check that expression, localization, and dimerization of our receptor/co-receptor system was functional. To achieve this, we first constructed two main types of our PRRs:

1. The PRR fused to YFP, so we can check correct expression and localization using fluorescence measurements and microscopy.
2. The PRR fused to the NanoLuc SmallBit, and the BAK1 co-receptor fused to NanoLuc LargeBit. These two parts are then co-expressed in *S. cerevisiae* to test dimerization using luminescence measurements.

## Constructs with YFP

1. [BBa\_K3610030](http://parts.igem.org/Part%3ABBa_K3610030): BAK1 with native signal peptide / YFP (BAK+)
2. [BBa\_K3610031](http://parts.igem.org/Part%3ABBa_K3610031): BAK1 without native signal peptide / YFP (BAK-)
3. [BBa\_K3610032](http://parts.igem.org/Part%3ABBa_K3610032): BAK1 ectodomain (eBAK)
4. [BBa\_K3610045](http://parts.igem.org/Part%3ABBa_K3610045): EFR ectodomain (eEFR)

First, we measured fluorescence levels in *S. cerevisiae* samples that had been transformed with these constructs and compared them to an untransformed control group. We measured fluorescence in a plate reader assay, and also using a flow cytometer. Both experiments confirmed higher fluorescence levels in the transformed samples than in the control, indicating that the constructs are expressed correctly.

![](https://static.igem.org/mediawiki/2020/a/a7/T--UZurich--1014.png)

*Fluorescence levels of samples with YFP constructs; Bars in yellow, green, blue and violet show expression of listed parts higher than the control.*

![](https://static.igem.org/mediawiki/2020/0/02/T--UZurich--facs2.png)

*Similar measurement done in a flow cytometer; violin charts show fluorescence intensity measured in 200000 cells per sample.*

We then imaged the transformed samples with a confocal microscope to see if the constructs are correctly localized at the cell membrane. The membrane was stained with FM4-64 to make the membrane more visible. eEFR, eBAK and BAK- (which all have the yeast alpha factor signal peptide) all show correct localization at the membrane in some cells, although BAK-, which retained the kinase domain, also showed up in what seemed like the vacuoles of the yeast cells. BAK+ still has the signal peptide from *A. thaliana* and clearly demonstrates that this is not sufficient for correct trafficking to the cell membrane.

![](https://static.igem.org/mediawiki/2020/1/1d/T--UZurich--image-2.png)

*[BBa\_K3610030 (BAK+)](http://parts.igem.org/Part%3ABBa_K3610030)
BAK+ shows moderate YFP fluorescence which is not co-localized with FM4-64, indicating that it is not localized at the membrane*

![](https://static.igem.org/mediawiki/2020/8/81/T--UZurich--image-3.png)

*[BBa\_K3610031 (BAK-)](http://parts.igem.org/Part%3ABBa_K3610031)
BAK- shows strong YFP fluorescence with little co-localization with FM4-64, indicating that some gets trafficked to the membrane*

![](https://static.igem.org/mediawiki/2020/c/ce/T--UZurich--image-4.png)

*[BBa\_K3610032 (eBAK)](http://parts.igem.org/Part%3ABBa_K3610032)
Most cells show strong fluorescence, clear co-localization with FM4-64 in a ring structure in about 1% of cells*

![](https://static.igem.org/mediawiki/2020/1/1c/T--UZurich--image-5.png)

*[BBa\_K3610045 (eEFR)](http://parts.igem.org/Part%3ABBa_K3610045)
Co-localization at the membrane in about 25% of cells. Dense clumps in the cell indicate that many constructs still get stuck*

From the plate reader and FACS results, we could see that our YFP constructs are expressed as expected. From the localization imaging results, we learned that the alpha-factor signal peptide is necessary to have correct localization at the cell membrane. In addition, the kinase domain also seems to play an important role in cellular trafficking, as all constructs which retained the kinase domain ended up, for the most part, inside of the cellular vacuole instead of the membrane.

## Constructs with NanoLuc

1. [BBa\_K3610038](http://parts.igem.org/Part%3ABBa_K3610038): BAK1 ectodomain fused to NanoLuc LargeBit (eEFR)
2. [BBa\_K3610043](http://parts.igem.org/Part%3ABBa_K3610043): EFR ectodomain fused to NanoLuc SmallBit (eBAK)

After confirming good expression and localization, we assembled the composite parts with the split NanoLuc attached to test receptor/co-receptor dimerization. These two parts were then coexpressed in *S. cerevisiae*. With both receptor and co-receptor present at the cell membrane, addition of the EFR elicitor elf18 should drive dimerization and increase activity of the split NanoLuc system, resulting in higher luminescence. We performed multiple dimerization assays under varying conditions and test parameters.

![](https://static.igem.org/mediawiki/2020/e/ef/T--UZurich--dimer3.png)

*Dimerization assay with eEFR ([BBa\_K3610038](http://parts.igem.org/Part%3ABBa_K3610038)) and eBAK ([BBa\_K3610043](http://parts.igem.org/Part%3ABBa_K3610043)). Shown are luminescence levels 30 min after addition of no epitope or elf18*

In summary, the split parts were correctly expressed, since the transformed sample showed much higher luminescence levels than the untransformed control. We were very happy to see the NanoLuc system working in action.
However, the dimerization - and with it NanoLuc activity - did not seem to be driven by the detection of the elf18 epitope, the ligand for EFR. We are not sure why that is the case. In plants, PRRs have helper proteins that suppress dimerization when no ligand is present. The absence of these factors might explain the high activity in the absence of the elicitor in our trials.

## Split NanoLuc characterization & improvement

We successfully used NanoLuc LargeBit ([BBa\_K1761005](http://parts.igem.org/Part%3ABBa_K1761005)), and created a codon optimized version for *S. cerevisiae* ([BBa\_K3610013](http://parts.igem.org/Part%3ABBa_K3610013)). These were then used in two composite parts:

1. [BBa\_K3610054](http://parts.igem.org/Part%3ABBa_K3610054): FKBP / LargeBit NanoLuc
2. [BBa\_K3610055](http://parts.igem.org/Part%3ABBa_K3610055): FKBP / LargeBit NanoLuc for S. cerevisiae

These parts where then each coexpressed with FRB / SmallBit NanoLuc ([BBa\_K3610056](http://parts.igem.org/Part%3ABBa_K3610056)) to test if the codon-optimized version would result in increased luminescence levels. A dimerization assay was performed with a plate reader by adding the NanoLuc subtrate and and Rapamycin to the samples.

![](https://static.igem.org/mediawiki/2020/2/24/T--UZurich--improv2.png)

*Luminescence levels of codon-optimized and normal NanoLuc LargeBit with and without Rapamycin*

The sample using the codon-optimized version of LargeBit showed higher luminescence levels.

##### *These results and more is documented in detail on our Results page.*

[Results](https://2020.igem.org/Team%3AUZurich/Results)

![](https://static.igem.org/mediawiki/2020/2/2c/T--UZurich--logo.png)

### UZurich 2020

[Home](https://2020.igem.org/Team%3AUZurich)
[Description](https://2020.igem.org/Team%3AUZurich/Description)
 [Results](https://2020.igem.org/Team%3AUZurich/Results)
[Team](https://2020.igem.org/Team%3AUZurich/Team)
[Human Practices](https://2020.igem.org/Team%3AUZurich/Human_Practices)
[Parts](https://2020.igem.org/Team%3AUZurich/Parts)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:UZurich/Experiments

iGEM UZurich 2020

[ ]

[* Home](https://2020.igem.org/Team%3AUZurich)

---

[* Medal Requirements](https://2020.igem.org/Team%3AUZurich/Medal)

---

* Project
[* - Description](https://2020.igem.org/Team%3AUZurich/Description)
[* - Design](https://2020.igem.org/Team%3AUZurich/Design)
[* - Engineering Success](https://2020.igem.org/Team%3AUZurich/Engineering)
[* - Results](https://2020.igem.org/Team%3AUZurich/Results)
[* - Implementation](https://2020.igem.org/Team%3AUZurich/Implementation)
[* - Contribution](https://2020.igem.org/Team%3AUZurich/Contribution)

---

* Team
[* - Members](https://2020.igem.org/Team%3AUZurich/Team)
[* - Attributions](https://2020.igem.org/Team%3AUZurich/Attributions)
[* - Collaborations](https://2020.igem.org/Team%3AUZurich/Collaborations)

---

* Human Practices
[* - Integrated Human Practices](https://2020.igem.org/Team%3AUZurich/Human_Practices)
[* - Education](https://2020.igem.org/Team%3AUZurich/Education)

---

[* Best Plant Synthetic Biology](https://2020.igem.org/Team%3AUZurich/Plant)

---

[* Parts](https://2020.igem.org/Team%3AUZurich/Parts)

---

* Lab
[* - Safety](https://2020.igem.org/Team%3AUZurich/Safety)
[* - Notebook](https://2020.igem.org/Team%3AUZurich/Notebook)
[* - Experiments](https://2020.igem.org/Team%3AUZurich/Experiments)

---

[* Sponsors](https://2020.igem.org/Team%3AUZurich/Sponsors)

### [iGEM UZurich 2020](https://2020.igem.org/Team%3AUZurich)

# Experiments

## Our Lab Protocols

We used the Benchling notebook software to document our daily progress in the lab and to have protocols for all of our regular
tasks and experiments in one central location. That way, any team member that was in the lab on a given day could access them easily,
making our work more efficient and less time-comsuming. Benchling also made it easy to export these protocols as PDFs and present them here.

---

[Diagnostic Restriction Digestion](https://static.igem.org/mediawiki/2020/3/3d/T--UZurich--protocol-DRD.pdf)

[Digestion Ligation](https://static.igem.org/mediawiki/2020/a/ab/T--UZurich--protocol-diglig.pdf)

[Dimerization assays](https://static.igem.org/mediawiki/2020/b/b7/T--UZurich--protocol-dimerization.pdf)

[Gel for Electrophoresis](https://static.igem.org/mediawiki/2020/8/80/T--UZurich--protocol-gel.pdf)

[Gel purification](https://static.igem.org/mediawiki/2020/e/e4/T--UZurich--protocol-gel_purification.pdf)

[In-Fusion cloning](https://static.igem.org/mediawiki/2020/a/a6/T--UZurich--protocol-in_fusion.pdf)

[E.Z.N.A. Plasmid DNA Mini Kit](https://static.igem.org/mediawiki/2020/e/e6/T--UZurich--protocol-miniprep.pdf)

[Fresh TAE loading gel stock solution](https://static.igem.org/mediawiki/2020/6/62/T--UZurich--protocol-TAE.pdf)

[OD600 measurement](https://static.igem.org/mediawiki/2020/d/d2/T--UZurich--protocol-od600.pdf)

[PCR & Colony PCR](https://static.igem.org/mediawiki/2020/1/18/T--UZurich--protocol-pcr.pdf)

[PCR product purification with Sepharose](https://static.igem.org/mediawiki/2020/1/1c/T--UZurich--protocol-sepharose.pdf)

[PCR product purification with Cycle Pure Kit](https://static.igem.org/mediawiki/2020/0/0b/T--UZurich--protocol-cycle_pure.pdf)

[E. coli Transformation](https://static.igem.org/mediawiki/2020/6/66/T--UZurich--protocol-transformation.pdf)

[PureYield Plasmid miniprep](https://static.igem.org/mediawiki/2020/6/63/T--UZurich--protocol-pure_yield.pdf)

[Resuspend IDT DNA](https://static.igem.org/mediawiki/2020/c/c8/T--UZurich--protocol-idt.pdf)

[TAP Media](https://static.igem.org/mediawiki/2020/a/ac/T--UZurich--protocol-tap.pdf)

[UZH in-house sequencing](https://static.igem.org/mediawiki/2020/0/0a/T--UZurich--protocol-sequencing.pdf)

[Western blotting](https://static.igem.org/mediawiki/2020/2/2d/T--UZurich--protocol-westernblot.pdf)

[Yeast Co-IP](https://static.igem.org/mediawiki/2020/0/01/T--UZurich--protocol-co-ip.pdf)

[Yeast transformation](https://static.igem.org/mediawiki/2020/7/7c/T--UZurich--protocol-yeast_transformation.pdf)

[Yeast transformation stock solutions](https://static.igem.org/mediawiki/2020/2/2d/T--UZurich--protocol-transformation-media.pdf)

[Yeast YNB -TRP dropout medium](https://static.igem.org/mediawiki/2020/a/a9/T--UZurich--protocol-trp.pdf)

[Yeast YPD medium](https://static.igem.org/mediawiki/2020/c/c9/T--UZurich--protocol-YPD.pdf)

![](https://static.igem.org/mediawiki/2020/2/2c/T--UZurich--logo.png)

### UZurich 2020

[Home](https://2020.igem.org/Team%3AUZurich)
[Description](https://2020.igem.org/Team%3AUZurich/Description)
 [Results](https://2020.igem.org/Team%3AUZurich/Results)
[Team](https://2020.igem.org/Team%3AUZurich/Team)
[Human Practices](https://2020.igem.org/Team%3AUZurich/Human_Practices)
[Parts](https://2020.igem.org/Team%3AUZurich/Parts)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:UZurich/Results

iGEM UZurich 2020

[ ]

[* Home](https://2020.igem.org/Team%3AUZurich)

---

[* Medal Requirements](https://2020.igem.org/Team%3AUZurich/Medal)

---

* Project
[* - Description](https://2020.igem.org/Team%3AUZurich/Description)
[* - Design](https://2020.igem.org/Team%3AUZurich/Design)
[* - Engineering Success](https://2020.igem.org/Team%3AUZurich/Engineering)
[* - Results](https://2020.igem.org/Team%3AUZurich/Results)
[* - Implementation](https://2020.igem.org/Team%3AUZurich/Implementation)
[* - Contribution](https://2020.igem.org/Team%3AUZurich/Contribution)

---

* Team
[* - Members](https://2020.igem.org/Team%3AUZurich/Team)
[* - Attributions](https://2020.igem.org/Team%3AUZurich/Attributions)
[* - Collaborations](https://2020.igem.org/Team%3AUZurich/Collaborations)

---

* Human Practices
[* - Integrated Human Practices](https://2020.igem.org/Team%3AUZurich/Human_Practices)
[* - Education](https://2020.igem.org/Team%3AUZurich/Education)

---

[* Best Plant Synthetic Biology](https://2020.igem.org/Team%3AUZurich/Plant)

---

[* Parts](https://2020.igem.org/Team%3AUZurich/Parts)

---

* Lab
[* - Safety](https://2020.igem.org/Team%3AUZurich/Safety)
[* - Notebook](https://2020.igem.org/Team%3AUZurich/Notebook)
[* - Experiments](https://2020.igem.org/Team%3AUZurich/Experiments)

---

[* Sponsors](https://2020.igem.org/Team%3AUZurich/Sponsors)

### [iGEM UZurich 2020](https://2020.igem.org/Team%3AUZurich)

# Results

## Expression and localization of the receptors in *S. cerevisiae*

Our first goal was to test whether the chassis would express the unaltered receptor and also whether it would localize the protein at the cell membrane.
Thus, we expressed the receptor together with a yellow fluorescent protein at the C-terminus of the protein to make expression visible.

Regarding the localization at the plasma membrane, it was at this point not clear if localization required a signal sequence native to *S. cerevisiae* or if the original signal peptide from the plant PRR would be functional in this organism. We created two versions of the constructs to test this, one with the original signal sequence at the N-terminus and one with the signal sequence from the alpha-mating Factor, a secreted pheromone from *S. cerevisiae.*

After transformation of *S. cerevisiae* cells and selection for successfully transformed cells on a synthetic dropout medium, we examined the cell cultures with multiple methods to get a better coverage of information about the expression of our constructs in the chassis. We first measured the fluorescence intensities of the cells with a plate reader and with a flow cytometer to examine whether expressing our constructs would lead to an increase in fluorescence with the wavelengths of YFP. While assessing fluorescence intensities is very helpful, its obvious limitations lie in its inability to localize the source of fluorescence within the cells. With this in mind, we also examined our cell cultures with confocal fluorescence microscopy. This approach allowed us to visualize YFP expression, while simultaneously examining the localization of fluorescence within the cells.

This was done for the full length receptors and for the receptor ectodomains alike. We managed to assemble the constructs with YFP for the BAK1 full length receptors with and without the native signal peptide (BAK+, [BBa\_K3610030](http://parts.igem.org/Part%3ABBa_K3610030) and BAK-, [BBa\_K3610031](http://parts.igem.org/Part%3ABBa_K3610031)), the BAK1 ectodomain (eBAK, [BBa\_K3610032](http://parts.igem.org/Part%3ABBa_K3610032)), the EFR ectodomain (eEFR, [BBa\_K3610045](http://parts.igem.org/Part%3ABBa_K3610045)) and the ectodomain of the receptor CORE (eCORE, [BBa\_K3610046](http://parts.igem.org/Part%3ABBa_K3610046)). FLS2 unfortunately could not be cloned correctly into an entry vector and thus had to be dropped.

#### Expression

In order to assess expression of our constructs, fluorescence was measured with a plate reader. When detecting and quantifying the fluorescence of different samples (λEx : 485 nm, λEm : 530 nm), transformed *S. cerevisiae* cells showed considerably higher fluorescence levels than the untransformed control, showing expression of our constructs fused to YFP in the transformed cells. Samples were measured with three biological replicates per construct.

![](https://static.igem.org/mediawiki/2020/a/a7/T--UZurich--1014.png)

*Biological triplicates per construct with technical triplicates per sample, normalized for OD600 and zero referenced for autofluorescence levels of blank sample. Note: columns ‘CORE’ and ‘EFR’ refer to the ectodomain-only version of the receptors*

It has been important to us to examine a sample with different approaches simultaneously, which is why we were eager to also measure fluorescence intensity by flow cytometry. In a first phase, 100,000 cells were measured from each biological replicate (488/530 FITC channel in a BD FACSCanto II flow cytometer).
In the next phase, the biological replicates for each construct were pooled together and 200,000 cells from each sample were measured.

![](https://static.igem.org/mediawiki/2020/3/3c/T--UZurich--results2.png)

*Left: Distribution of fluoresence intensity in 4 samples per construct. Right: Pooled samples for each construct, showing distribution of fluoresence intensity. All except eCORE show higher intensity.*

The measurements made with the flow cytometer confirmed what had been observed previously with the plate reader. With the exception of the construct with eCORE, cells transfected with our constructs showed considerably higher overall fluorescence intensities than the negative control. This observation provides further evidence for expression of our constructs in *S. cerevisiae*.

#### Localization

After evaluating the results from the fluorometer we examined the yeast samples with confocal fluorescence microscopy. We examined cells for YFP fluorescence (λEx = 514 nm, λEx = 527 nm) and in addition to that, cells were stained with FM4-64, a dye which stains the plasma membrane but gets quickly endocytosed, which is why one may see vesicles stained with FM4-64. If the constructs get expressed in *S. cerevisiae*, we should see a strong increase in yellow fluorescence. If the constructs also get localized at the plasma membrane (PM), we should be able to observe colocalization of YFP and the FM4-64 stain.

![](https://static.igem.org/mediawiki/2020/7/7a/T--UZurich--image-1.png)

*Figure 1: Untransformed *S. cerevisiae*
A: YFP, B: FM4-64, C: light field, D: merged
Untransformed samples show very little fluorescence in the YFP spectrum*

![](https://static.igem.org/mediawiki/2020/1/1d/T--UZurich--image-2.png)

*Figure 2: [BBa\_K3610030](http://parts.igem.org/Part%3ABBa_K3610030) (BAK+)
BAK+ shows moderate YFP fluorescence which is not co-localized with FM4-64, indicating that it is not localized at the membrane*

![](https://static.igem.org/mediawiki/2020/8/81/T--UZurich--image-3.png)

*Figure 3: [BBa\_K3610031](http://parts.igem.org/Part%3ABBa_K3610031) (BAK-)
BAK- shows strong YFP fluorescence with little co-localization with FM4-64, indicating that some gets trafficked to the membrane*

![](https://static.igem.org/mediawiki/2020/c/ce/T--UZurich--image-4.png)

*Figure 4: [BBa\_K3610032](http://parts.igem.org/Part%3ABBa_K3610032) (eBAK)
Most cells show strong fluorescence, clear co-localization with FM4-64 in a ring structure in about 1% of cells*

![](https://static.igem.org/mediawiki/2020/1/1c/T--UZurich--image-5.png)

*Figure 5: [BBa\_K3610045](http://parts.igem.org/Part%3ABBa_K3610045) (eEFR)
Co-localization at the membrane in about 25% of cells. Dense clumps in the cell indicate that many constructs still get stuck*

![](https://static.igem.org/mediawiki/2020/5/5d/T--UZurich--image-6.png)

*Figure 6: [BBa\_K3610046](http://parts.igem.org/Part%3ABBa_K3610046) (eCORE)
Cells show weak fluorescence situated in the vacuoles rather than the membrane*

What we observed with the microscope was in alignment with our expectations after evaluating the results from the fluorometric analysis. Cell transfected with the receptor ectodomain of eCORE did not show increased fluorescence levels when examined with a plate reader. Similarly, fluorescence microscopy also indicated very weak yellow fluorescence and no localization at the cell membrane was visible.
BAK1+ cells did show an increase in fluorescence when examined with the fluorometer, but to a much lower degree when compared to the constructs containing the signal peptide from *S. cerevisiae* (with the exception of eCORE). The same observation was made under the microscope. BAK+ cells showed increased fluorescence which was not co-localized with the FM4-64 stain, indicating that this construct did not get integrated into the plasma membrane.

The other three constructs which were examined with the confocal microscope, the BAK1 ectodomain (eBAK, [BBa\_K3610032](http://parts.igem.org/Part%3ABBa_K3610032)), the BAK1 receptor with the signal peptide from *S. cerevisiae* (BAK-, [BBa\_K3610031](http://parts.igem.org/Part%3ABBa_K3610031)) and the EFR ectodomain (eEFR, [BBa\_K3610045](http://parts.igem.org/Part%3ABBa_K3610045)), showed much stronger fluorescence. This was the case when they were examined with a plate reader and with the fluorescent microscope.
Additionally, cells transfected with plasmids containing the eBAK and eEFR show clear localization of our constructs at the cell membrane. The proportion of the cells which display co-localization with FM4-64, however, varies strongly. In BAK- cells, the overlap between the yellow fluorescence and the membrane stain is rather scarce, but nevertheless observable, which indicates that some of it gets trafficked to the cell periphery.
eBAK cells did show high fluorescence and in about 1% of the cells a very clear ring structure was visible at the cell membrane. eEFR cells were of similar appearance. In this case about 25% of the cells had clearly localized the construct at the plasma membrane.

The results from the plate reader and the confocal fluorescence microscopy suggest that our constructs, the ectodomain of BAK1 and EFR and the full length receptor BAK1 with the signal sequence from the alpha-Factor are actually expressed in *S. cerevisiae*. BAK1 with the native signal peptide is expressed as well and eCORE is probably expressed at very low levels. Additionally, microscopy imaging suggested that the eEFR and eBAK are localized at the cell membrane. This localization at the cell periphery is a big success as it shows that the signal peptide from the alpha-Mating factor did, in fact, induce translation into the membrane. Additionally, it was shown that the intracellular kinase domain is not necessary for membrane localization, but much rather caused the construct to get stuck in what seems to be the cellular vacuoles.
Having achieved expression of these PRRs in *S. cerevisiae* and demonstrated that they get integrated into the plasma membrane opens the doors to many future applications of PRRs in this organism.

## Dimerization of the receptors in *S. cerevisiae*

For our system to be working two things are required. The receptor and co-receptor must be able to interact, enabling them to dimerize, leading to a reconstitution of NanoBit parts to catalyze the luminescence-accompanied reaction of furmazine to furimamide. The dimerization, further, must be driven by the epitope to which the receptor is specific.

#### *Testing dimerization using split NanoLuc*

To test this, we transformed *S. cerevisiae* to express both a receptor - eCORE ([BBa\_K3610051](http://parts.igem.org/Part%3ABBa_K3610051)) or eEFR ([BBa\_K3610043](http://parts.igem.org/Part%3ABBa_K3610043)), each fused to NanoLuc SmallBiT - and the co-receptor eBAK1 ([BBa\_K3610038](http://parts.igem.org/Part%3ABBa_K3610038)) fused to NanoLuc LargeBit at the same time.

In a first assay, the substrate furimazine and the bacterial elicitors were added to the samples and then incubated for 30 minutes. After this incubation with substrate and the bacterial epitopes, the samples were examined with a luminescence reader. All samples were brought to the same optical density (OD600 = 0.34).
The results of the luminescence assay showed increased luminescence when samples were examined which had previously been transfected with the plasmids containing the constructs, especially when the *S. cerevisiae* cells had been transfected with plasmids containing the eEFR construct. The results, however, suggest that the dimerization of the NanoLuc parts was not driven by ligand-dependent interaction of the receptors.

![](https://static.igem.org/mediawiki/2020/7/7a/T--UZurich--dimer1.png)

*Dimerization assay with eEFR/eCORE and eBAK. Shown are luminescence levels 30min after addition of no epitope, elf18 or csp22*

To get more data, a second assay was performed, this time only with eEFR samples.
In the second assay, the luminescence levels were measured immediately after addition of the bacterial elicitors, elf18 or csp22, and the substrate furimazine. All samples were brought to the same optical density (OD600 = 0.26).

![](https://static.igem.org/mediawiki/2020/5/5c/T--UZurich--dimer2.png)

*Dimerization assay with eEFR and eBAK. Shown are luminescence levels directly after addition of furimazin and no epitope, elf18 or csp22*

The assay was repeated a third time. This time, the substrate was added to the samples and then they were incubated for 30 minutes, after which the bacterial elicitor was added, this time only elf18, and the samples were measured directly. The samples were, again, resuspended in order to keep them at the same OD.

![](https://static.igem.org/mediawiki/2020/e/ef/T--UZurich--dimer3.png)

*Dimerization assay with eEFR and eBAK. Shown are luminescence levels 30 min after incubating the samples in Nanoluc substrate*

Multiple assays have been performed with the NanoBit constructs. Each time, the LargeBit and SmallBit parts were able to dimerize to a functional NanoLuc protein, which was made evident by an increase in luminescence after addition of furimazine.
Addition of bacterial epitope did not further increase the levels of luminescence, which indicates that the interaction between the NanoBits is not driven by ligand-dependent interaction of the receptors. It therefore seems that the bacterial epitope elf18 does not induce an interaction between BAK1 and EFR when they are expressed in *S. cerevisiae*. This, however, needs further examination.

#### *Testing dimerization using Co-Immunoprecipitation*

Another way to test for dimerization is Co-Immunoprecipitation

Beads were treated with anti-FLAG antibodies. To these beads, whole cell lysate of yeast cells with eBAK::LargeBiT and eEFR::SmallBiT was added. One sample has been treated with elf18, while the other was not. This was to investigate if elf18 induces dimerization. The eEFR contains the FLAG tag and is thus expected to bind to the AB-treated beads. If eBAK interacts with eEFR upon exposure to elf 18, it should attach itself to the beads by binding to eEFR. All proteins which attach themselves can then be eluted from the beads and visualised via Western blotting. eEFR is visualized with anti-FLAG-HRP and eBAK with anti-HA-HRP. There are six different protein samples which get visualized via western blotting:

* Input mock, which is untreated whole cell lysate
* Unbound mock, which is the fraction of the untreated whole cell lysate which didn’t attach to the AB-treated beads
* Input elf18, which is whole cell lysate which has been treated with elf18
* Unbound elf18, which is the fraction of the whole cell lysate treated with elf 18 which didn’t attach to the AB-treated beads
* IP unbound, which is the fraction of the untreated whole cell lysate which attached to the AB-treated beads
* IP elf18, which is the fraction of the elf 18 treated whole cell lysate which attached to the AB-treated beads

![](https://static.igem.org/mediawiki/2020/6/62/T--UZurich--coip.jpeg)

Unfortunately the blot which should have visualized eBAK1 doesn’t show any clear bands, thus making it impossible to determine if dimerisation occured. What we can see is the blot which visualises eEFR. Interestingly, there are 3 distinct bands in each lane, suggesting that eEFR exists in 3 different forms of eEFR with different weights. This could be caused by post translational modification like glycosylation. We see that the lowest band at around 70 kDa appears weaker in the column with the unbound cell lysate compared to the input, suggesting that eEFR attaches itself to the beads treated with anti-FLAG as expected. This band at 70 kDA then again shows up in the columns containing the fraction which was eluted from the beads. Interestingly, there are also two thick bands above 70 kDa in these eluent columns, which doesn’t really make sense, as we would expect to see a difference in the thickness of these bands between the columns containing the whole cell lysate and the flow-through, but there the bands seem relatively similar.

#### *Testing dimerization using split mCherry*

To ensure we could test our approach of a system which would generate an output with a split-protein system, even if we had failed to assemble our constructs with the NanoLuc protein, we aimed at having a system put to test with a different reporter protein. We chose to use mCherry.

The activity of these constructs were assessed in the same manner as the ones using NanoLuc. A fluorescence assay was performed (λEX = 587nm and λEM = 610nm), using samples of the same optical density (OD600 = 0.5).

![](https://static.igem.org/mediawiki/2020/a/a4/T--UZurich--mcherry.png)

*Dimerization assay of samples cotransformed with eEFR fused to split-mCherry ([BBa\_K3610034](http://parts.igem.org/Part%3ABBa_K3610034)) and eBAK fused to split-mCherry ([BBa\_K3610040](http://parts.igem.org/Part%3ABBa_K3610040))*

In this case, presence of the bacterial elicitor elf18 seems to increase fluorescence. Since this is, however, the case for for both yeasts containing our plasmid and untransformed yeast, we cannot conclude that this has been achieved through ligand-dependent receptor dimerization, especially when considering that the fluorescence levels were not increased when transfected *S. cerevisiae* cells were examined, in fact, there was no significant difference between the untransformed control and the samples with cells containing the plasmids with our constructs.
It is clear that this deserves further examination. We propose to repeat the measurements to increase the data coverage and rule out errors during pipetting or labelling the samples. It should further be attempted to lower autofluorescence for yeast cells that resemble the fluorescence of mCherry.

## Part Improvement: Codon-optimize NanoLuc LargeBit for *S. cerevisiae*

In order to further characterize the split-NanoLuc system, we compared the luminescence levels of a system using the LargeBit sequence from the registry ([BBa\_K1761005](http://parts.igem.org/Part%3ABBa_K1761005)) with a system using a new sequence which had been codon optimized for expression in *S. cerevisiae* ([BBa\_K3610014](http://parts.igem.org/Part%3ABBa_K3610014)).
In order to drive the interaction between the two NanoBits the two sequences were fused to the sequences encoding FKBP12 and FRB, which are rapamycin ligands that are used to drive protein-protein interaction.
Plasmids containing the constructs FRB:SmallBit and FKBP12:LargeBit, were used to transfect *S. cerevisiae* cells. This was done twice. In one case, the LargeBit sequence was codon optimized for expression in yeast ([BBa\_K3610055](http://parts.igem.org/Part%3ABBa_K3610055)), while one yeast culture was transfected with plasmids containing the original sequence ([BBa\_K3610054](http://parts.igem.org/Part%3ABBa_K3610054)).

![](https://static.igem.org/mediawiki/2020/a/a5/T--UZurich--improv1.png)

![](https://static.igem.org/mediawiki/2020/2/24/T--UZurich--improv2.png)

*Both: Luminescence levels of codon-optimized and normal NanoLuc LargeBit with and without Rapamycin. The codon-optimized version shows slightly higher luminescence.*

Initial comparison of the average luminescent output measured by the plate reader indicated similar levels of expression. A increased luminescence could be detected when the codon optimized sequence was used.

## Further steps

Our experiments using the NanoBiT® system, while showing that some dimerization occurs between BAK1::LBit+EFR::Sbit, showed no response to the elicitor of EFR, elf18, which makes it difficult to adapt this system into a usable biosensor as is. This absence of a response might be due to the absence of negative regulators which are always present in plants together with the PRRs. These negative regulators are called BAK1-interacting-receptor-like kinases 2 and 3 (BIR 2 and 3) which bind to BAK1 in the absence of elicitors [1]. The coexpression of BIR 2 and 3 might be a promising attempt to reduce the amount of signal in the absence of elicitors.

As we have seen during confocal imaging, the absence or presence of the kinase domain on the receptors plays a major role in the localisation of the receptors. We have seen that only a small fraction of yeast cells expressing BAK1 without kinase domain localise said receptor on the plasma membrane. This means that only a few cells co-localise eBAK1 and eEFR, which reduces the potential for dimerization. It would certainly be worthwhile to investigate the trafficking of full length EFR and to see if it localises inside the vacuoles, like full length BAK1. Then it needs to be investigated if dimerization can be induced by elf18 when these receptors are co-localised inside of the vacuole instead of the plasma membrane.

It might also just be that the receptors don’t get expressed correctly in yeast, either due to incorrect folding or other post-translational processes, like glycosylation. This is why a future team working on this project should focus on expressing our constructs in *Chlamydomonas reinhardtii*, something we were very keen on doing, but for which we, unfortunately, didn’t have any time.

#### Expanding our construct design to increase modularity

If we manage to get a visual output in response to the elicitors, we can think about building upon our design to make them more modular. When using constructs which rely on the complementation of fluorescent or chemiluminescent proteins, we are inherently limited by the amount of these proteins available, as finding the correct split site in a protein is no trivial task. In addition, the constructs that we designed up until now should deliver a visual output very quickly after exposure to an elicitor, but the strength of the output is heavily dependent on the amount of receptors in the plasma membrane. If the output of one receptor pair is quite weak, it would require a greater amount of receptors to be expressed, which might place a metabolic burden on our chassis. This is why we thought about a system which would induce a signal cascade upon dimerization instead of an immediate visual output, leading to the expression of a desired reporter protein. We found that the NTHU Formosa team developed a system which would be applicable for this vision. They utilized nanobodies, or split antibodies, situated in the cell membrane which would dimerize upon exposure to a specific antibody [2]. They attached two fragments of the split tobacco etch virus (TEV) protease to these Nanobodies. Upon dimerization, the TEV protease would cut the TEV cutting site which is attached to one half of the split TEV protease, which would then release a transcription factor that initiates expression of a reporter. We think that this system holds great potential to be adapted in combination with our receptors, as these nanobodies act quite similarly to our PRRs.

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[1] Couto, D. and Zipfel, C., 2016. Regulation of pattern recognition receptor signalling in plants. Nature Reviews Immunology, 16(9), p.537
[2] https://2018.igem.org/Team:NTHU\_Formosa

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### UZurich 2020

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