iGEM 2019 — Team Tuebingen

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# Team:Tuebingen/Notebook

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GLP.exe - Notebook

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##### GLP.exe](https://2019.igem.org/Team%3ATuebingen)

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# Notebook

##### Week 1 (08 Jul - 12 Jul)

**08 Jakob Patrick Eva** After moving into our new lab and settling in, first
practical
steps like aliquoting our primers or preparing LB-Medium and LB-Agar were taken.
**09 Jakob Patrick Eva** We amplified the cas3 gene out of *E. coli*
gDNA by PCR
(Q5 Polymerase) and transformed the biobricks BBa\_K608351 (K2), BBa\_K091001
(K5),
BBa\_I13453 (K10), BBa\_K584000 (AraC Promotor), BBa\_K117008 (LsrR Promotor),
BBa\_R0073 (Mnt Promotor) in competent *E. coli* DH5-Alpha (NEB iGEM Kit).
**10 Eva Patrick** We amplified cascade genes out of *E. coli* MG1655
gDNA via PCR
(Q5 Polymerase) and checked the amplification of cas3 and cascade via agarose
gel electrophoresis. (Fig. 1) We picked colonies for all but the Ba\_K608351
(K2) transformations (no growth) for inoculating overnight cultures and made
a new batch of competent *E. coli* DH5-Alpha cells. **11 Jakob Eva
Patrick**
By Miniprep (Qiagen) we extracted the DNA of the overnight cultures.
Amplification via PCR (Q5 Polymerase) of cascade was repeated (Fig. 2).
BBa\_K608351 (K2) was transformed again and the competent cells tested.

[![A generic square placeholder](https://static.igem.org/mediawiki/2019/a/a8/T--Tuebingen--Gel1.png)](https://static.igem.org/mediawiki/2019/a/a8/T--Tuebingen--Gel1.png "Fig. 1. 17/10/19 Gel elctrophoresis of cas3 and cascade
                                            PCR")

Figure 1: 17/10/19 Gel
elctrophoresis of cas3 and cascade
PCR. Gel loaded (from left to right): Ladder 1kb, cas3 (72°C), cas3 (68°C),
H2O, cascade (72°C), cascade (68°C), H2O. It shows positive PCR of cas3
(68°C), expected at ~2.7 kb.

[![A generic square placeholder image](https://static.igem.org/mediawiki/2019/2/2a/T--Tuebingen--Gel2.png)](https://static.igem.org/mediawiki/2019/2/2a/T--Tuebingen--Gel2.png "Fig. 2. 07/11/19 Gel electrophoresis of cascade PCR")

Figure 2: 07/11/19 Gel
electrophoresis of cascade PCR.
Gel loaded (from left to right): Ladder 1kb, cascade (68°C), cascade (64°C).
It shows positive PCR of cas3 (68°C), expected at ~4.4 kb.

##### Week 2 (15 Jul - 19 Jul)

**15 Jakob Patrick Eva Luzi** Transformations of DH5-Alpha cells with the
biobricks
were repeated. 16 Patrick, Eva Since there repeatedly weren’t any colonies
for BBa\_K608351 (K2), we transformed NovaBlue™ competent cells with this
biobrick. Overnight cultures of the other biobricks as well as an overnight
culture of *E. coli* Nissle were inoculated. **17 Eva Patrick
Katharina** Glycerol
stocks of the overnight cultures were prepared. Competent *E. coli* Nissle
cells
were produced and the growth of *E. coli* Nissle was analysed
preliminarily.
After Miniprep (Qiagen) of BBa\_K516030 (rfp) a PCR (Pfu Polymerase) was run.
After digestion of the PCR products cas3 and cascade as well as the plasmid
BBa\_I13453 (K10), K10\_cascade and K10\_cas3 were ligated. **18 Eva Patrick**
The
K10\_cascade and K10\_cas3 ligations were digested (single) and analysed by gel
electrophoresis, together with the rfp PCR product (Fig. 3). The gel did not
show a result for K10\_cascade and K10\_cas3. rfp (PCR Product) and BBa\_R0073
(Mnt Promotor) were digested and ligated. **19 Eva Patrick** Digestion and
Ligation
of cascade, cas3 and K10 were repeated with increased amounts of DNA. DH5-Alpha
cells were transformed with K10\_cascade, K10\_cas3 and BBa\_R0073(Mnt)\_rfp

[![A generic square placeholder](https://static.igem.org/mediawiki/2019/3/30/T--Tuebingen--Gel3.png)](https://static.igem.org/mediawiki/2019/3/30/T--Tuebingen--Gel3.png "Fig. 3. 07/18/19 Gelelctrophoresis of rfp PCR, digests
                                            of K10_cascade and K10_cas3")

Figure 3: 07/18/19
Gelelctrophoresis of rfp PCR, digests
of K10\_cascade and K10\_cas3. Gel loaded (from left to right): Ladder 1
kb, rfp 1, rfp 2, rfp 3, K10\_cas3 (no heat inact.), K10\_cas3, K10\_cascade,
Ladder 1 kb. It shows positive PCR of rfp, expected at ~890 bp. No result
for digestions.

##### Week 3 (22 Jul - 26 Jul)

**23 Eva Jakob Patrick** Preparation of LB-Medium, LB-Agar and
Chloramphenicol plates.
Overnight cultures for K10\_cascade, K10\_cas3 and BBa\_R0073(Mnt)\_rfp colonies
were
inoculated. After double digestion of BBa\_K584000 (AraC Promoter), BBa\_K117008
(LsrR Promotor),
the fragments ParaC and PLsrR were extracted from the gel. **24 Eva Patrick**
After Miniprep of
K10\_cascade, K10\_cas3 and BBa\_R0073(Mnt)\_rfp (Qiagen) the plasmids were single
digested. Gel
electrophoresis showed the empty backbone (Fig. 4). The isolated fragments ParaC
and PLsrR were
ligated into the backbone pSB1K3 after digestion. pSB1K3\_ParaC and pSB1K3\_PLsrR
were transformed
in DH5-Alpha cells. **25 Eva** Overnight cultures for pSB1K3\_ParaC and
pSB1K3\_PLsrR colonies were
inoculated. **26 Eva** After Miniprep (Qiagen) of pSB1K3\_ParaC and
pSB1K3\_PLsrR, the plasmids were
digested. Gel electrophoresis showed successful ligations (Fig. 5).

[![A generic square placeholder](https://static.igem.org/mediawiki/2019/1/13/T--Tuebingen--Gel4.png)](https://static.igem.org/mediawiki/2019/1/13/T--Tuebingen--Gel4.png "Fig. 4. 07/24/19 Single digestions after Miniprep")

Figure 4: 07/24/19 Single
digestions after Miniprep.
Gel loaded (from left to right): Ladder 1kb, BBa\_R0073(Mnt)\_rfp 1.1,
BBa\_R0073(Mnt)\_rfp 1.2, K10\_cas3 1.1-3.2, K10\_cascade 1.1-3.2.
BBa\_R0073(Mnt)\_rfp expected at ~3 kb, K10\_cas3 expected at ~4.9 kb,
K10\_cascade expected at ~6.6 kb.

[![A generic square placeholder image](https://static.igem.org/mediawiki/2019/b/b6/T--Tuebingen--Gel5.png)](https://static.igem.org/mediawiki/2019/b/b6/T--Tuebingen--Gel5.png "Fig. 5. 07/26/19 Single Digestions after Miniprep")

Figure 5: 07/26/19 Single
Digestions after Miniprep.
Gel loaded (from left to right): pSB1K3\_ParaC 1-8, pSB1K3\_PLsrR 1-8, Ladder
1kb, pSB1K3 2.2-2.3, 3.1-3.2. Bands of ~3 kb suggest successful ligations.

##### Week 4 (29 Jul - 02 Aug)

**30 Patrick, Katharina** The team moved into a new laboratory. The
synthesized
sequences (K0, K3, K4, K6, K7, K15) arrived and were aliquoted. Chloramphenicol
plates were prepared. cascade, cas3, BBa\_I13453 (K10), rfp and BBa\_R0073
(Mnt Promotor) digestions and ligations were repeated and transformed.
**31 Patrick Jakob** Overnight cultures for K10\_cas3 colonies were
inoculated,
there was no growth of the other transformations. A PCR (Pfu polymerase) was
run on the synthesized constructs, it wasn’t successful. The Minipreps of
pSB1K3\_ParaC and pSB1K3\_PLsrR were used for retransformation. **01 Patrick**
The
K10\_cas3 overnight culture was miniprepped. Overnight cultures for pSB1K3\_ParaC,
pSB1K3\_PLsrR, as well as K10\_cascade and BBa\_R0073(Mnt)\_rfp (some late colonies
grew)
were inoculated. **02 Patrick** Glycerol stocks for pSB1K3\_ParaC were
prepared,
there was no growth in pSB1K3\_PLsrR overnight cultures. After Miniprep of
rpSB1K3\_ParaC, K10\_cascade and BBa\_R0073(Mnt)\_rfp the plasmids (also K10\_cas3)
were digested and a gel electrophoresis was run. It showed the transformation
of empty backbone in K10\_cascade and BBa\_R0073(Mnt)\_rfp, successful ligation for
pSB1K3\_ParaC and unsuccessful ligation for K10\_cas3 (Fig. 6).

[![A generic square placeholder](https://static.igem.org/mediawiki/2019/8/82/T--Tuebingen--Gel6.png)](https://static.igem.org/mediawiki/2019/8/82/T--Tuebingen--Gel6.png "Fig. 6. 08/02/19 Digestions after Miniprep")

Figure 6: 08/02/19
Digestions after Miniprep. Gel loaded
(from left to right): Ladder 1 kb, K10\_cas3 1-10, K10\_cascade 1-4,
BBa\_R0073(Mnt)\_rfp 1-2, positive control, pSB1K3\_ParaC. Negative results
for K10\_cas3, expected at ~4.9 kb, and K10\_cascade, expected at 6.6 kb.
Successful ligation of pSB1K3\_ParaC, ~ 3 kb.

##### Week 5 (05 Aug - 09 Aug)

**05 Patrick Eva Antonia** PCR (Q5 polymerase) and PCR (Paq polymerase) was
run on the
synthesized constructs, they weren’t successful. cascade and cas3 were
successfully
amplified via PCR(Q5). **06 Eva Patrick Antonia** A gradient PCR (Q5) was run
on the constructs.
It was discovered that the currently used agarose was falsely prepared with
water instead
of TAE-Buffer and was discarded. cascade, cas3 and BBa\_I13453 (K10) were
digested and ligated.
**07 Patrick Eva Luzi Antonia** A new PCR (Q5) was run on the synthesized
constructs,
gel electrophoresis suggested positive amplification (Fig. 7). By amplification
with accordingly designed primer, K0 has been added a TetR promoter and is now
the construct K1. Due to a technical malfunction in the building further work
was delayed.

[![A generic square placeholder](https://static.igem.org/mediawiki/2019/4/46/T--Tuebingen--Gel7.png)](https://static.igem.org/mediawiki/2019/4/46/T--Tuebingen--Gel7.png "Fig. 7. 08/08/19 PCR of synthesized constructs")

Figure 7: 08/08/19 PCR of
synthesized constructs.
Gel loaded (from left to right): Ladder 1kb, K1 (expected at 660bp),
K3 (expected at 1 kb), K4 (expected at 1 kb), K6 (expected at 1.5 kb),
K7 (expected at 700 bp), K15 (expected at 1 kb).

##### Week 6 (12 Aug - 16 Aug)

**12 Patrick Katharina** After respective digestion and DNA clean up,
pSBC13\_K1,
pSBC13\_K3, pSBC13\_K6, K5\_K4, K10\_cas3, K10\_cascade and BBa\_R0073(Mnt)\_rfp were
ligated. **13 Patrick Eva Antonia Marie** DH5-Alpha cells were transformed
with
Yesterday’s ligations. **14 Luzie Marie Eva** A colony PCR (Taq polymerase)
was
run on pSBC13\_K1, but the gel electrophoresis was negative (Fig. 8). Overnight
cultures of pSBC13\_K1, pSBC13\_K3, pSBC13\_K6, K5\_K4, K10\_cas3 and K10\_cascade
colonies were inoculated. **15 Marie Patrick Eva** After Miniprep the
plasmids were
double digested. Gel electrophoresis suggested successful ligations of
pSBC13\_K6,
K10\_cas3, K10\_cascade and BBa\_R0073(Mnt)\_rfp (Fig. 9). **16 Patrick**
Miniprep of
pSBC13\_K1 was digested but gel electrophoresis was negative. cascade and cas3
were amplified by PCR (Q5 polymerase). The synthesized construct K2 arrived,
was diluted and amplified via PCR (Q5 polymerase).

[![A generic square placeholder](https://static.igem.org/mediawiki/2019/2/25/T--Tuebingen--Gel8.png)](https://static.igem.org/mediawiki/2019/2/25/T--Tuebingen--Gel8.png "Fig. 8. 08/14/19 Colony PCR of pSB1C3_K1 transformation")

Figure 8: 08/14/19 Colony
PCR of pSB1C3\_K1 transformation.
Gel loaded (from left to right): Ladder 1kb, pSB1C3\_K0 1-10.

[![A generic square placeholder image](https://static.igem.org/mediawiki/2019/c/cc/T--Tuebingen--Gel9.png)](https://static.igem.org/mediawiki/2019/c/cc/T--Tuebingen--Gel9.png "Fig. 9. 08/15/19 Digestions after Miniprep")

Figure 9: 08/15/19
Digestions after Miniprep. Gel loaded
(from left to right): Ladder 1kb, pSB1C3\_K6 1, empty, pSB1C3\_K6 2, pSB1C3\_K3
1-2,
K10\_cas3 1-2, K10\_cascade 1-2, BBa\_R0073(Mnt)\_rfp 1-4.
K6 expected at ~2 kb and 1.5 kb, K3 expected at ~2 kb and 1 kb, cas3
expected at ~2.4 kb, ~2 kb and ~0.3 kb, cascade
expected at ~5.1 kb, ~1,2 kb and ~0.3 kb, rfp expected at ~2 kb and ~1 kb.

##### Week 7 (19 Aug - 23 Aug)

**19 Patrick Jakob Eva** After respective digestion and DNA clean up,
pSB1C3\_K1,
pSB1C3\_K3, pSB1C3\_K7 and K5\_K4 were ligated. The BBa\_R0073(Mnt)\_rfp plasmid was
digested to isolate PMnt\_rfp by gel extraction (Fig. 10). The backbone pSB1K3
was
digested with respective enzymes and pSB1K3\_PMnt\_rfp was ligated. K1, K2, K3,
K4,
K7 and K15 were amplified via PCR (Q5 polymerase) with a new programm. **20 Eva
Patrick**
Last week’s pSBC13\_K6, K10\_cas3 and K10\_cascade were sent off to Sanger
sequencing.
DH5-Alpha cells were transformed with yesterday’s ligations as well as
retransformed
with K10\_cascade . **21 Marie Zoe Patrick** A new batch of competent cells
was prepared
and overnight cultures of pSB1C3\_K1, pSB1C3\_K7, K5\_K4 and K10\_cascade
inoculated.
**22 Patrick Eva** Sanger Sequencing was positive on K10\_cascade.
Chloramphenicol and
Kanamycin plates were prepared. After Miniprep of pSB1C3\_K1, pSB1C3\_K7, K5\_K4
and
K10\_cascade the plasmids were digested and run on a gel. Digestions of pSB1C3\_K1
and K5\_K4 were negative. The new competent cells were transformed with
pSB1C3\_K3,
pSB1K3\_PMnt\_rfp and iGEM competent cell test plasmids. **23 Marie Patrick**
The
constructs K1, K2, K3, K4, K7 and K15 were amplified by PCR (Q5 polymerase).
Double digestion of pSB1C3\_K7 and K10\_cascade was repeated. The gel
electrophoresis
suggested successful ligation of K7, the result for K10\_cascade was unclear
(Fig. 11).

[![A generic square placeholder](https://static.igem.org/mediawiki/2019/7/77/T--Tuebingen--Gel10.png)](https://static.igem.org/mediawiki/2019/7/77/T--Tuebingen--Gel10.png "Fig. 10. 08/19/19 Digestion of BBa_R0073(Mnt)_rfp for
                                            gel extraction")

Figure 10: 08/19/19
Digestion of BBa\_R0073(Mnt)\_rfp for
gel extraction. Gel loaded (from left to right): Ladder 1 kb,
BBa\_R0073(Mnt)\_rfp.
Desired fragment at ~0.5 kb.

[![A generic square placeholder image](https://static.igem.org/mediawiki/2019/9/92/T--Tuebingen--Gel11.png)](https://static.igem.org/mediawiki/2019/9/92/T--Tuebingen--Gel11.png "Fig. 11. Digestion after Miniprep")

Figure 11: Digestion after
Miniprep. Gel loaded (from left to right):
Ladder 1 kb, pSB1C3\_K7 1-2, K10\_cascade 1-5, K10\_cas3.

##### Week 8 (26 Aug - 30 Aug)

**26 Patrick** A new PCR (Q5 polymerase) was run on the constructs and the
successfully
amplified DNA purified. K10\_cascade and pSB1C3\_K6 were retransformed.
Inoculation of
overnight cultures for K10\_cascade and K10\_cas3 old colonies. **27 Patrick Eva
Jakob Antonia**
Miniprep of K10\_cascade and K10\_cas3 followed by digestion and gel
electrophoresis,
which suggested successful ligation of K10\_cascade, K10\_cas3 is XY (Fig. 12).
All synthesized constructs (K0-K15) were digested and ligated with their
respective
backbone. Overnight cultures of K10\_cascade and pSB1C3\_K6 retransformations were
inoculated. **28 Patrick Eva Antonia** The BBa\_R0073(Mnt)\_rfp plasmid was
digested to
isolate PMnt\_rfp and the backbone pSB1K3 was digested with respective enzymes.
pSB1K3\_PMnt\_rfp was ligated. DH5-Alpha cells were transformed with yesterday’s
construct (K0-K15) ligations. Overnight cultures of K10\_cascade and pSB1C3\_K6
were
miniprepped and digested. The gel electrophoresis showed bands suggesting a
successful ligation of K10\_cascade and pSB1C3\_K6 (Fig. 13). The synthesized
constructs were amplified via PCR (Q5 polymerase) for our Experimenta
collaboration.
**29 Eva Antonia Patrick** A colony PCR (Q5) was run on K5\_K4, the other
transformations didn’t show growth. Gel electrophoresis of the PCR didn’t
show a positive result. To amplify pSB1C3, a biobrick of the iGEM distribution
plates was diluted and a PCR (Q5 polymerase) was run. The constructs (K0-K15)
were digested. K0, K2, K3, K15 were ligated with their backbone pSB1C3. K4 was
ligated with BBa\_K091001 (K5) and K7 was ligated with pSB1C3\_K6. The work on the
cell penetrating peptides (CPPs) was started by amplifying gfp and synb1 via PCR
(Q5 polymerase) (Fig. CPP 1). Primer were dissolved to 100 µM. **30 Antonia Eva
Katharina**
The colony PCR for K4\_K5 was repeated, but remained negative. PCR (Q5
polymerase)
was run on CPPs. Gel electrophoreses of the PCR showed double bands.

[![Figure 12](https://static.igem.org/mediawiki/2019/3/39/T--Tuebingen--Gel12.png)](https://static.igem.org/mediawiki/2019/3/39/T--Tuebingen--Gel12.png "Figure 12")

Figure 12: 08/27/19
Digestions after Miniprep. Gel loaded
(from left to right): ladder 1 kb, K10\_cascade 1-7, K10\_cas3 1-7.
K10\_cascade
expected at ~6.6 kb, K10\_cas3 expected at ~4.9 kb.

[![Figure 13](https://static.igem.org/mediawiki/2019/5/55/T--Tuebingen--Gel13.png)](https://static.igem.org/mediawiki/2019/5/55/T--Tuebingen--Gel13.png "Figure 13")

Figure 13: 08/28/19
Digestions after Miniprep. Gel loaded
(from left to right): Ladder 1 kb, K10\_cascade 1, empty, K10\_cascade 2-4,
pSB1C3\_K6
1-4. K10\_cascade expected at ~5.1 kb, ~1,2 kb and ~0.3 kb, pSB1C3\_K6
expected at ~ 2 kb
and ~1.6 kb.

##### Week 9 (02 Sep - 06 Sep)

**02 Lina Antonia Luzi Jakob Eva Famke** As preliminary testing, a growth
curve of
*E. coli* Nissle at 37°C, pH 7, was generated. DH5-Alpha cells were
transformed
with the ligations of constructs (K0-K15) as well as pSB1K3\_PMnt\_rfp and the
distribution kit biobrick. The CPPs tat-lk15, tat, yta2, tp10, pVec and
penetratin
were amplified by PCR (HotStart Polymerase). The gel electrophoresis showed
double
bands (Fig. CPP 2). **03/04 Lina** Growth curves of *E. coli* Nissle
(EcN) for different
temperatures were created. EcN cultures were inoculated with an overnight
culture
and grown for 120 minutes at 37°C. Next, temperature was changed to 0°C, 4°C,
8°C
and 25°C, while one control was kept at 37°C. 37°C and 8°C were chosen for
sample
preparation for RNA-Seq. EcN was grown 120 min, after being inoculated from one
overnight culture, at pH7. Then, media was changed to the respective pH (6, 5,
4, 3, 2, 1)
and growth was observed. Growth in pH4 was replicated. EcN was grown in
different
concentrations of Hydrogen Peroxide (1 µM, 10 µM, 25 µM, 100 µM), infused after
two hours of growth at 37°C, pH7 in LB medium. EcN, inoculated from the same
overnight
culture, were grown under four anaerobic conditions (LB, mGAM anaerobic,
mGAM+1.3
mM Metformin, mGAM+0,25mM cholic acid) at 37°C, pH7. EcN aerobic growth in LB
was
used as control. Samples for RNA sequencing were taken after 270 minutes for all
anaerobic conditions, since the increase of pressure within the flasks indicated
a
change of metabolism of EcN to fermentation processes. Aerobic LB growth samples
were taken at OD600=1.0.

EcN, inoculated from the same overnight culture, were grown in mGAM medium under
anaerobic conditions at 37°C, pH 7 supplemented with either 50% supernatant of
different species or 50% of water as a control for depleted nutrients.
Supernatant of Bacteroides
thetaiotaomicron, Prevotella copri, Ruminococcus gnavus, Bifidobacterium
adolescentis and
Clostridium difficile was previously produced by Dr. Lisa Maier. Bacteroides
samples were
taken for RNA sequencing after 225 minutes. EcN were lastly grown in the growth
medium provided by
iGEM Stuttgart, in comparison to LB medium. Overnight cultures were used for
inoculation of three samples with 5ml each and OD600 was measured every 45 mins.
For the dryfreezed samples, 9 µL skim milk were added to 1 µg wet weight of
bacteria,
frozen at -80°C for 2 h, 42 h at -20°C and 2h at 25°C. Afterwards they were
resuspended in LB.

All OD600 measurements were conducted with the Nanospectrometre by Implen. For
the
results check [Project/
Nissle](https://2019.igem.org/Team%3ATuebingen/Nissle).
**04 Eva Antonia** A batch of competent cells was prepared. pSB1C3\_K0,
pSB1C3\_K2,
pSB1C3\_K3, pSB1C3\_K15, pSB1C3\_K6\_K7, K5\_K4 and pSB1K3\_PMnt\_rfp colonies were
analyzed
by colony PCR(Q5). Gel electrophoresis didn’t show positive result. **05 Eva
Antonia**
Further gel electrophoresis of yesterday’s colony PCR remained negative.
Digestions and ligations of constructs were repeated. Overnight cultures of
distribution kit biobrick colonies were inoculated. **06 Marie** Miniprep of
distribution kit biobrick.

[![Figure 14](https://static.igem.org/mediawiki/2019/8/88/T--Tuebingen--GelCPP2.png)](https://static.igem.org/mediawiki/2019/8/88/T--Tuebingen--GelCPP2.png "GelCPP2")

Figure CPP 2: 09/02/19 PCR
of CPP parts. Gel loaded
(from left to right): Ladder 1 kb, pVec (expected at ~100 bp), tat
(expected at ~70 bp), tat-lk15 (expected at ~120 bp), tp10 (expected at ~90
bp),
yta2 (expected at ~90 bp).

##### Week 10 (09 Sep - 13 Sep)

**10 Eva Antonia** K0, K2, K3, K4, K6, K7, K15 were amplified via PCR
(Q5 polymerase) using primers designed for Gibson assembly, the PCR didn’t work.
After digestion of the CPPs and their backbones, the plasmids pET-28b(+)\_synb1
as well as pMiniT2.0\_pVec and pMiniT2.0\_tat-lk15 were ligated. **11 Eva
Antonia**
A gradient PCR of the constructs was run with Gibson primers, gel
electrophoresis
suggested successful amplification of K1, K2 and K15 (Fig. 14). **12 Eva
Antonia**
Gibson-PCR of K3, K4, K6, and K7 was successfully repeated. For Gibson assembly
the backbones pSB1C3 and BBa\_K091001 (K5) were single digested. pSB1C3\_K1\_K15,
pSB1C3\_K2\_K3, K5\_K4 and pSB1C3\_K6\_K7 were assembled and transformed.
Additionally,
pET-28b(+)\_synb1 was transformed. **13 Eva Antonia** The transformations
didn’t
show colonies so the Gibson assembly was repeated and transformed with provided
NovaBlue™ competent cells. Colonies for pET-28b(+) were picked and an
over-weekend
culture (RT) inoculated. The ligations of pMiniT2.0\_pVec and pMiniT2.0\_tat-lk15
were transformed in DH5-Alpha cells.

[![Figure 14](https://static.igem.org/mediawiki/2019/4/45/T--Tuebingen--Gel14.png)](https://static.igem.org/mediawiki/2019/4/45/T--Tuebingen--Gel14.png "Figure 14")

Figure 14: 09/11/19 Gibson
PCR. Gel loaded (from left to right): Ladder 1 kb, empty, K15 1-3, K1 1-2,
empty, K1 3-5, K2 1-5. K15 expected at ~1 kb, K1 expected at ~0.5 kb, K2
expected at ~1 kb.

##### Week 11 (16 Sep - 20 Sep)

**16 Antonia** The Novablue™ Gibson transformation showed comprehensive
growth so they were diluted by streaking. The over-weekend cultures of
pET-28b(+)\_synb1 were miniprepped. For pMiniT2.0\_pVec and pMiniT2.0\_tat-lk15
colonies were picked and overnight cultures inoculated. **17 Marie Eva
Antonia**
The BBa\_R0073(Mnt)\_rfp plasmid was digested to isolate PMnt\_rfp and the backbone
pSB1K3 was digested with respective enzymes. pSB1K3\_PMnt\_rfp was ligated.
The dilution streaks of Novablue™ Gibson transformations were diluted again.
**18 Marie Eva** pSB1K3\_PMnt\_rfp was transformed. A colony PCR was run on
Novablue™
Gibson Assembly colonies. Gel electrophoresis was negative. **19 Marie
Antonia**
The colony PCR was repeated but remained negative. DH5-Alpha cells were
transformed
with pMiniT2.0\_cpp (tat, yta2, tp10, penetratin). A miniprep of pET-28b(+) was
performed. The product was controlled by gel electrophoresis but was negative
(Fig. CPP 3). **20 Marie Antonia** The BBa\_R0073(Mnt)\_rfp plasmid was
digested to
isolate PMn\_rfp and the backbone pSB1K3 was digested with respective enzymes.
pSB1K3\_PMnt\_rfp was ligated. synb1 was amplified by a PCR. A colony PCR was
performed to control the transformed pMiniT2.0\_cpp plasmids. Over-weekend
cultures (RT) of Novablue™ Gibson Assembly colonies were inoculated.

[![Figure 14](https://static.igem.org/mediawiki/2019/b/b7/T--Tuebingen--GelCPP3.png)](https://static.igem.org/mediawiki/2019/b/b7/T--Tuebingen--GelCPP3.png "Figure 14")

Figure CPP3: 09/19/19
Miniprep of pET-28b(+)\_synb1. Gel loaded (from left to right): Ladder 1 kb,
pET-28b(+)\_synb1 1, pET-28b(+)\_synb1 2, pET-28b(+)\_synb1 3.

##### Week 12 (23 Sep - 27 Sep)

**23 Marie Antonia** Transformation of pSB1K3\_PMnt\_rfp. The over-weekend
cultures
of Gibson assembly were used for a new colony PCR, lysing the cells before the
reaction.
It remained negative. pET-28b(+) vector was digested and ligated with synb1.
The pET-28b(+)\_synb1 was transformed. **24 Marie Eva** Overnight cultures of
pSB1K3\_PMnt\_rfp
colonies were inoculated. Colony-PCR of pMiniT2.0\_cpp was performed, gel
electrophoresis
showed double bands (Fig. CPP 4). Overnight a ligation of pET-28b(+) and synb1
was performed.
**25 Marie Antonia** Nanodrop measuring of Gibson-PCR product. Transformation
of pET-28b(+)\_synb1.
After Miniprep of pSB1K3\_PMnt\_rfp the plasmids were digested. Gel
electrophoresis suggested
ligation of the empty backbone. Single digestion of pSB1C3 and BBa\_K091001 (K5)
followed by
Gibson assembly was repeated and DH5-Alpha cells transformed. **27 Antonia
Marie** No growth of
new Gibson assembly. Over-weekend (RT) cultures of Novablue™ Gibson Assembly
colonies were
inoculated. Cultures of pMiniT2.0\_penetratin and pET-28b(+)\_synb1 colonies were
inoculated.

[![Figure 14](https://static.igem.org/mediawiki/2019/6/6b/T--Tuebingen--GelCPP4.png)](https://static.igem.org/mediawiki/2019/6/6b/T--Tuebingen--GelCPP4.png "Figure 14")

Figure CPP 4: 09/24/19 Gel
electrophoresis of Colony PCR.
Gel loaded (from left to right): Ladder 1 kb, penetratin 1, penetratin 2,
tat 1,
tat 2, YTA-2 1, YTA-2 2, tp10 1, TP 10 2. Bands expected at ~100 bp.

##### Week 13 (30 Sep - 04 Oct)

**30 Marie Lina Antonia** The BBa\_R0073(Mnt)\_rfp plasmid was digested to
isolate
PMnt\_rfp and the backbone pSB1K3 was digested with respective enzymes.
pSB1K3\_PMnt\_rfp was ligated. Miniprep of Novablue™ Gibson Assembly
over-weekend cultures. pSB1C3 was amplified via PCR (Taq polymerase).
Transformation of BBa\_K592024 (bfp) from the iGEM distribution Kit and
inoculation of K2 (BBa\_K608351) on plate. Repeating of colony-PCR of
pMiniT2.0\_penetratin, pMiniT2.0\_tat, pMiniT2.0\_yta2, pMiniT2.0\_tp10 colonies.
**01 Marie Lina** Antonia Digestion of Gibson assembly Minipreps and gel
electrophoresis,
which was negative (Fig. 15). Overnight cultures for K2 (BBa\_K608351)
were inoculated. Newly synthesized cpp+gfp parts arrived. Amplification
tp10+gfp, penetratin+gfp, yta2+gfp, pVec+gfp and synb1+gfp by PCR (Fig. CPP 5).
**02 Lina Antonia Jakob** Miniprep of K2 overnight cultures and digestion.
Double digestion of BBa\_K091001 (K5) and clean up of the digested backbone.
pSB1C3 and the constructs (K0-K15) were amplified via PCR (Taq polymerase).
BBa\_K575011 (Ev1), BBa\_K523001 (Ev2), BBa\_K763001 (Ev3), BBa\_K763002 (Ev4) and
BBa\_K398108 (Ev5) from the iGEM distribution kit were transformed in DH5-Alpha
cells.
Amplification of CPP PM r10 (~100 bp) (Fig. CPP 6). Digest of CPPs. (Fig. CPP6).
Retransformation of pET-28b(+). **04 Jakob Lina Antonia** Double Digestion of
pSB1C3 followed by clean up and Gibson assembly. DH5-Alpha cells were
transformed with pSB1C3\_K0\_K15, pSB1C3\_K2\_K3, K5\_K4 and pSB1C3\_K6\_K7.
Restriction digestion and ligation of pSB1C3\_K0, pSB1C3\_K2, pSB1C3\_K3,
K5\_K4, pSB1C3\_K6, pSB1C3\_K7 and pSB1C3\_K15 were repeated and transformation
performed. Over-weekend (RT) cultures for the distribution kit biobricks were
inoculated. Amplification of new CPP : PM penetratin (Fig. CPP 7).

[![Figure 15](https://static.igem.org/mediawiki/2019/a/a6/T--Tuebingen--Gel15.png)](https://static.igem.org/mediawiki/2019/a/a6/T--Tuebingen--Gel15.png "Figure 15")

Figure 15: 10/01/19
Digestion after Miniprep
(Gibson Assembly). Gel loaded (from left to right): Ladder 1 kb,
pSB1C3\_K1\_K15 1-2,
pSB1C3\_K2\_K3 1-2, K5\_K4 1-2, pSB1C3\_K6\_K7 1-2, empty, empty, Miniprep
pSB1C3\_K6\_K7 undigested.

[![Figure 15](https://static.igem.org/mediawiki/2019/7/75/T--Tuebingen--GelCPP5.png)](https://static.igem.org/mediawiki/2019/7/75/T--Tuebingen--GelCPP5.png "GelCPP5")

Figure CPP 5: 10/01/19 PCR
of CPP parts. Gel loaded (from left to right): Ladder 1 kb,
tp10+gfp, penetratin+gfp, yta2+gfp, pVec+gfp, synb1+gfp. All expected at
900kb.

[![Figure 15](https://static.igem.org/mediawiki/2019/b/b4/T--Tuebingen--GelCPP6.png)](https://static.igem.org/mediawiki/2019/b/b4/T--Tuebingen--GelCPP6.png "GelCPP5")

Figure CPP 6: 10/02/19
Amplification of PM r10 with XbaI primer. (expected at 100 bp). Gel loaded
(from left to right): r10 1.0, r10 2.0, r10 3.0.

[![Figure 15](https://static.igem.org/mediawiki/2019/2/2b/T--Tuebingen--GelCPP7.png)](https://static.igem.org/mediawiki/2019/2/2b/T--Tuebingen--GelCPP7.png "GelCPP5")

Fig. CPP 7. 10/02/19
Digest (expected at 100 bp). Gel
loaded (from left to right): penetratin+gfp, penetratin+gfp (clean up),
tp10+gfp, tp10+gfp (clean up), pVec+gfp, pVec+gfp (clean up), synb1+gfp,
synb1+gfp (clean up), yta2+gfp, yta2+gfp (clean up), pET-28b(+), pET-28b(+)
XbaI, pET-28b(+) EcoRI

##### Week 14 (07 Oct - 13 Oct)

**07 Jakob Lina Antonia** Overnight cultures of pSB1C3\_K0\_K15 (Gibson),
K5\_K4(Gibson),
K5\_K4 and pSB1C3\_K7 were inoculated. Miniprep of Ev1-Ev5 over-weekend cultures.
Transformation of BBa\_K132018, BBa\_K098991, BBa\_K876004 and BBa\_K74100X from
iGEM distribution kit. **08 Antonia** Miniprep of construct overnight
cultures was
conducted, followed by single digest, which suggested unsuccessful ligations.
**09 Lina** Digest of PM r10 CPP and pET-28b(+) (Fig. CPP 8). **12 Steffen
Famke Antonia**
The characterisation of BBa\_K398108 (Ev5), a salt tolerant cluster, was
proceeded
after identity of the plasmid was confirmed via digestion (Fig. 16). An
overnight
culture of the bacteria (from which the Ev5 mini originated), as well as
DH5alpha
wild type cells were inoculated in LB and put on the shaker at 37°C. As a basis
for our modeling, the growth of *E. coli* Nissle 1917 under in minimal
media was to
be analysed. For this, an overnight culture was inoculated and M9 medium was
prepared.
**13 Lina Jakob Famke Antonia** The salt tolerance experiment was performed.
For this
LB 0M KCl, 0.05M KCl, 0.1M KCl, 0.2M KCl, 0.4M KCl and 0.8M KCl in LB were
prepared
and sterile filtered. The experiment was then performed in triplicates with
each DH5alpha wild type and the salt cluster bearing DH5alpha cells. Of each
100µL were inoculated in 15 ml of the respective medium. OD600 was measured
every 45min using the Nanophotometer of Implen. The experiment was finished
upon reaching the end of the exponential phase. The measured data (Fig. 17),
was logarithmized, nonlinearly fitted and normalised against each other (Fig.
18),
yielding the result that the salt tolerance cluster does not confer to increased
tolerance against KCl in *E. coli* DH5alpha. The minimal medium M9 was
inoculated
with EcN overnight cultures. Since calcium chloride did not dissolve properly,
one group of four samples in M9 was used with the unsolved. After several hours
of stirring, another group of M9 with solved calcium chloride was ready. Thus,
in total for each medium, 4 samples with 20 ml medium of either LB (control),
M9\_unsolved and M9\_solved were inoculated with 500 µL of EcN and OD600 was
measured every 45 min using the Nanophotometer of Implen. The measuring was
stopped upon the LB control entering the plateau phase (Fig. 19).

Please find the recipe for the M9 medium provided by Arie Geerlof -
Helmholtz Center Munich under the following link: <https://static.igem.org/mediawiki/2019/2/20/T--Tuebingen--M9_recipe.pdf>

[![Figure 16](https://static.igem.org/mediawiki/2019/2/22/T--Tuebingen--GelCPP8.png)](https://static.igem.org/mediawiki/2019/2/22/T--Tuebingen--GelCPP8.png "GelCPP8")

Figure CPP 8: 10/09/19
Digest of PM r10 CPP and pET-28b(+). Gel loaded (from left to right): PM r10
CPP, PM r10 CPP, pET-28b(+), pET-28b(+), pET-28b(+) NcoI, pET-28b(+) EcoRI

[![Figure 16](https://static.igem.org/mediawiki/2019/3/3d/T--Tuebingen--Gel16.png)](https://static.igem.org/mediawiki/2019/3/3d/T--Tuebingen--Gel16.png "Figure 16")

Figure 16:
10/12/19 Digestion after Miniprep. Gel loaded (from left to right):
Ladder 1 kb, BBa\_K575011 (Ev1) single digest, BBa\_K575011 (Ev1)
double digest, BBa\_K763002 (Ev4) single digest, BBa\_K763002 (Ev4)
double digest, BBa\_K398108 (Ev5) single digest, BBa\_K398108 (Ev5)
double digest.

[![Figure 16](https://static.igem.org/mediawiki/2019/e/eb/T--Tuebingen--figure17-white.png)](https://static.igem.org/mediawiki/2019/e/eb/T--Tuebingen--figure17-white.png "Fig. 17.  Mean of the triplicates and standard deviation of the measured growth curves. ")

Figure 17: Mean of
the triplicates and standard deviation of the measured growth
curves.

[![Figure 16](https://static.igem.org/mediawiki/2019/9/95/T--Tuebingen--salttolerance_data_final_white.png)](https://static.igem.org/mediawiki/2019/9/95/T--Tuebingen--salttolerance_data_final_white.png "Figure 18")

Figure 18: Growth rate
[h-1] and [%] growth of tolerant normalised to wt *E. coli*
DH5alpha.Fit was performed with the mean of the three samples.

[![Figure 16](https://static.igem.org/mediawiki/2019/9/98/T--Tuebingen--minimalmedium_white.png)](https://static.igem.org/mediawiki/2019/9/98/T--Tuebingen--minimalmedium_white.png "Fig. 19. Comparison of EcN growth in minimal medium (M9 solved/unsolved CaCl2) versus LB medium at pH7 and 37°C. ")

Figure 19: Comparison of
EcN growth in minimal medium
(M9 solved/unsolved CaCl2) versus LB medium at pH7 and 37°C.

### RNA-Seq

For the preparation of the cultures of *E. coli* Nissle 1917 Lina used
LB-Lennox medium. For one sample the pH was adjusted to pH 4 and was incubated
at 37°C. In another sample 100 µM H2O2 were added and incubated at 37°C. The
remaining three samples were incubated at different temperatures (8,25,37°C).

After pelleting the bacterial cultures the pellet was taken up in 1 ml RNAlater
to ensure that the RNA is not degraded and expression levels do not change.
Patrick and Lina isolated the RNA from the 24 samples with the PureLink RNA Mini
Kit by Thermo Fisher Scientific using 500ml of the diluted pellet. The isolated
nucleic acid was checked via an agarose gel and the amount estimated via Qubit
measurement before being treated with DNase I by Roche.

[![Placeholder](https://static.igem.org/mediawiki/2019/5/51/T--Tuebingen--NCCT-RNA-neu.png)](https://static.igem.org/mediawiki/2019/5/51/T--Tuebingen--NCCT-RNA-neu.png "Fig.20:Gel electrophoresis of the RNA after RNA isolation of each sample.")

Figure 20: Gel
electrophoresis of the RNA after RNA isolation of each sample. From left
to right: 1 kb Ladder, 4x LB aerobe 37°C, 4x LB aerobe 25°C, 4x LB
aerobe 8°C, 1 kb Ladder, 4x LB aerobe H2O2, 4x LB aerobe pH4, 4x LB
aerobe dry freeze. The two strong bands in each column represent the
rRNA which was depleted in further steps.

Patrick purified the RNA via the RNA Clean & Concentrator Kit by Zymo. Another
agarose gel was conducted and RNA amount estimated via Qubit. rRNA depletion was
conducted with the riboPOOL kit by siTOOLs, the RNA was again purified with the
RNA Clean & Concentrator Kit by Zymo and RNA amount estimated via Qubit
measurement. Despite very low RNA amount it was proceeded with the Library
preparation with the CORALL Total RNA-Seq Library Prep Kit by Lexogen. RNA
amounts were analysed via Qubit measurement and Library quality was analysed via
FemtoPulse measurement by Agilent. Additionally a qPCR of a pool of all 24
libraries was conducted to better determine the Library amount with the NEBNext
Library Quant Kit by NEB. Since the library quality was not sufficient, the rest
of the DNase I treated samples was used to start depletion and library
preparation again. This time the Total RNA Seq Library Prep Kit by Zymo was
used, RNA amount estimated via Qubit measurement and Library quality analysed
via FemtoPulse as well as a qPCR. Again RNA amount was low after depletion and
library preparation and library quality was not high enough for sequencing.
Jakob repeated the *E. coli* growth and the RNA Extraction with the Qiagen
RNeasy kit instead of the dry freezed samples the condition was changed to LB
aerob with pH8. The nucleic acid concentration was high after extraction and the
RNA showed no sign of degradation.

A DNAse digestion was conducted, the cDNA synthesis, the library preparation and
the rRNA depletion were done with the Universal Prokaryotic RNA-Seq Prokaryotic
AnyDeplete ™ kit from NuGEN. At a first glance, the library preparation seemed
successful. Soon, the pooled libraries will be loaded onto the Illumina MiSeq
System for sequencing.

[![Placeholder](https://static.igem.org/mediawiki/2019/4/45/T--Tuebingen--RNA-2-NCCT-jakob.png)](https://static.igem.org/mediawiki/2019/4/45/T--Tuebingen--RNA-2-NCCT-jakob.png "Fig.21: Gel electrophoresis of the RNA after RNA isolation of each sample.")

Figure 21: Gel
electrophoresis of the RNA after RNA isolation of each sample.
From left to right: 1 kb Ladder, 1x RNA before cleanup 4x LB aerobe H2O2, 4x
LB aerobe
8°C, 4x LB aerobe 25°C, 1x LB aerobe 37°C, 1 kb Ladder 1x RNA before cleanup
3x LB aerobe
37°C, 4x LB aerobe pH4, 4x Lb aerob pH8. The two strong bands in each column
represent
the rRNA which was depleted in further steps

Lina and Katharina prepared the anaerob bacterial cultures with the different
conditions: LB aerob, Lb anaerob, mGAM medium anaerob, mGAM medium with 1.3 mM
Metformin anaerob, mGAM medium with 0.25 mM cholic acid anaerob and mGAM medium
with the supernatant of a Bacteroides thataiotaomicron culture. Cultures were
again pelleted and taken up in RNAlater. Patrick isolated the RNA with the
PureLink RNA Mini Kit by Thermo Fisher Scientific and conducted a Qubit
measurement before and after treatment with DNaseI by Roche. Jakob and Steffen
tested the quality of the RNA via an Agilent RNA 6000 Nano Bioanalyzer.
Degradation was clearly visible but the samples could be used for the library
prep. The rRNA was depleted via the NEBNext (R) rRNA Depletion Kit. Thereafter,
our collaborators at the EMBL conducted the automated RNA library preparation
and sequencing.

---

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# Team:Tuebingen/Experiments

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# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Tuebingen/Results

![](https://static.igem.org/mediawiki/2019/4/49/T--Tuebingen--DNA_gif.gif)

GLP.exe - Results

[![logo](https://static.igem.org/mediawiki/2019/2/2a/T--Tuebingen--white_logo_animated.gif)

##### GLP.exe](https://2019.igem.org/Team%3ATuebingen)

* [OUR
  STORY](/Team%3ATuebingen/Story)
* PROJECT

  [Description](/Team%3ATuebingen/Description)
  [Kill Switch](/Team%3ATuebingen/Killswitch)
  [Nissle](/Team%3ATuebingen/Nissle)
  [Incretin](/Team%3ATuebingen/Incretin)
  [Model](/Team%3ATuebingen/Model)
  [Software](/Team%3ATuebingen/Software)
  [Results](/Team%3ATuebingen/Results)
  [Demonstrate](/Team%3ATuebingen/Demonstrate)
  [Notebook](/Team%3ATuebingen/Notebook)
  [Outlook](/Team%3ATuebingen/Outlook)
  [Entrepreneurship](/Team%3ATuebingen/Entrepreneurship)
* PARTS

  [Parts
  Overview](/Team%3ATuebingen/Parts)
  [Basic
  Parts](/Team%3ATuebingen/Basic_Part)
  [Improved
  Parts](/Team%3ATuebingen/Improved_Part)
  [Characterized
  Parts](/Team%3ATuebingen/Characterized_Part)
  [Composite
  Parts](/Team%3ATuebingen/Composite_Part)
  [Part
  Collection](/Team%3ATuebingen/Part_Collection)
  [Downloads](/Team%3ATuebingen/Downloads)
* PEOPLE

  [Team](/Team%3ATuebingen/Team)
  [Collaborations](/Team%3ATuebingen/Collaborations)
  [Sponsors](/Team%3ATuebingen/Sponsors)
  [Attributions](/Team%3ATuebingen/Attributions)
  [Gallery](/Team%3ATuebingen/Gallery)
* HUMAN PRACTICES

  [Overview](/Team%3ATuebingen/Human_Practices)
  [Education & Public Engagement](/Team%3ATuebingen/Public_Engagement)
  [Experts](/Team%3ATuebingen/Experts)
  [Sustainable Development Goals](/Team%3ATuebingen/SDG)
  [Experimenta
  Science Center](/Team%3ATuebingen/Experimenta)
  [Survey](/Team%3ATuebingen/Survey)
* AWARDS

  [Integrated Human Practise](/Team%3ATuebingen/Human_Practices)
  [Education & Public Engagement](/Team%3ATuebingen/Public_Engagement)
  [Supporting Entrepreneurship](/Team%3ATuebingen/Entrepreneurship)
  [Model](/Team%3ATuebingen/Model)
  [Software](/Team%3ATuebingen/Software)
* [SAFETY](/Team%3ATuebingen/Safety)
* [JUDGING FORM ⇗](https://igem.org/2019_Judging_Form?team=Tuebingen)

# Results

## Results

This year, our aim was to overcome the physical, emotional and financial burden of Type 2 Diabetes
mellitus. We targeted our objectives by designing a new, affordable therapy strategy, characterising
our chassis organism, doing in silico work, as well as developing a new biocontainment feature for
safe application of living organisms as therapeutics. Accordingly, we chose the tracks i) Therapeutics,
ii) Software and iii) New Application

## Therapeutics

### Targets reached:

* design and synthesis of a ready to express therapeutic
  ([Incretin](https://2019.igem.org/Team%3ATuebingen/Incretin),
  [Composite Part](https://2019.igem.org/Team%3ATuebingen/Composite_Part))
* evaluation of the public's perception of GMOs as therapeutics
  ([Survey](https://2019.igem.org/Team%3ATuebingen/Survey))
* education on GMOs and Diabetes prevention
  ([Public\_Engagement](https://2019.igem.org/Team%3ATuebingen/Public_Engagement), [Experimenta](https://2019.igem.org/Team%3ATuebingen/Experimenta))
* development of a viable business strategy for the entry of a live GMO into the market
  ([Entrepreneurship](https://2019.igem.org/Team%3ATuebingen/Entrepreneurship))
* design of a test format and the compounds to test the delivery of exendin-4 via cell penetrating peptides
  ([Software](https://2019.igem.org/Team%3ATuebingen/Software))
* characterisation and data generation on chassis organism *E. coli* Nissle 1917
  ([Nissle](https://2019.igem.org/Team%3ATuebingen/Nissle))

### Next steps:

* glucose-dependent expression of the therapeutic
  ([Incretin](https://2019.igem.org/Team%3ATuebingen/Incretin))
* proof of concept in vitro
  ([Outlook](https://2019.igem.org/Team%3ATuebingen/Outlook),
  [Incretin](https://2019.igem.org/Team%3ATuebingen/Incretin))
* integration of a biocontainment module
  ([KillSwitch](https://2019.igem.org/Team%3ATuebingen/Killswitch))
* designing features for the optimized growth of engineered *E. coli* Nissle 1917
  ([Entrepreneurship](https://2019.igem.org/Team%3ATuebingen/Entrepreneurship))

## New application of CRISPR/Cas3

### Targets reached:

* design and synthesis of all parts related to the CRISPR/Cas3 kill switch
  ([KillSwitch](https://2019.igem.org/Team%3ATuebingen/Killswitch),
  [Basic Part](https://2019.igem.org/Team%3ATuebingen/Basic_Part))
* isolation of Cas3 nuclease and the Cascade sequence
  ([KillSwitch](https://2019.igem.org/Team%3ATuebingen/Killswitch))
* cloning of the subunits of the kill switch
  ([Notebook](https://2019.igem.org/Team%3ATuebingen/Notebook))
* development of reporters for CRISPR-module activity
  ([Notebook](https://2019.igem.org/Team%3ATuebingen/Notebook))
* development of a design which allows the usage of live GMOs as therapeutics
  ([Human Practice](https://2019.igem.org/Team%3ATuebingen/Human_Practices),
  [KillSwitch](https://2019.igem.org/Team%3ATuebingen/Killswitch))

### Next steps:

* finalisation of the cloning of regulating modules
  ([Outlook](https://2019.igem.org/Team%3ATuebingen/Outlook),
  [KillSwitch](https://2019.igem.org/Team%3ATuebingen/Killswitch))
* testing of all modules via the reporters
* integration of the parts into one system
* proof of concept of the kill switch: degradation and killing of bacterium in prohibitive conditions
  ([Outlook](https://2019.igem.org/Team%3ATuebingen/Outlook))
* integration into the *E. coli* Nissle genome
  ([Outlook](https://2019.igem.org/Team%3ATuebingen/Outlook))
* assembly of the biocontainment system and the therapeutic modules
* experimental confirmation in vitro
  ([Outlook](https://2019.igem.org/Team%3ATuebingen/Outlook))
* providing a service to integrate our new biocontainment switch into other applications to allow for the safe use of live GMOs
  ([Entrepreneurship](https://2019.igem.org/Team%3ATuebingen/Entrepreneurship))

## Modelling

### Targets reached:

* Determined optimal growth medium for *E. coli* Nissle and validated it in the lab
  ([Model](https://2019.igem.org/Team%3ATuebingen/Model),
  [Nissle](https://2019.igem.org/Team%3ATuebingen/Nissle))
* Predicted interaction and competition of different gut microbe compositions
  ([Model](https://2019.igem.org/Team%3ATuebingen/Model))
* Examined and modelled *E. coli* Nissle transcriptomic response to various stress factors
  ([Nissle](https://2019.igem.org/Team%3ATuebingen/Nissle))

### Next steps:

* Analyze NCCT RNA-Seq dataset
* optimisation of our chassis organism *E. coli* Nissle and growth conditions according to the metabolic model
  ([Nissle](https://2019.igem.org/Team%3ATuebingen/Nissle),
  [Model](https://2019.igem.org/Team%3ATuebingen/Model),
  [Entrepreneurship](https://2019.igem.org/Team%3ATuebingen/Entrepreneurship))
* publication of data acquired during our characterisation
  ([Outlook](https://2019.igem.org/Team%3ATuebingen/Outlook))
* Extend the Nissle metabolic model with more reactions suitable for our engineered system and predict its interaction with the gut microbiome
  ([Model](https://2019.igem.org/Team%3ATuebingen/Model))

## Software

### Targets reached:

* Optimisation of our project by determining the superior penetrability of cell penetrating peptides by C3 Pred: Penetratin
  ([Software](https://2019.igem.org/Team%3ATuebingen/Software),
  [Incretin](https://2019.igem.org/Team%3ATuebingen/Incretin))
* Develop an easy to use interface and allow for the simple integration of our software into workflows
  ([Software](https://2019.igem.org/Team%3ATuebingen/Software))

### Next steps:

* Include additional training data to improve our model
* Improve cell penetrating peptide design support by automatically evaluating point mutations

## Conclusion

While we reached many subordinate targets of our project, we still require proof of concept for
the designs of our therapeutic and kill switch. Nonetheless, we demonstrated that our software
tool, as well as metabolic modeling, worked very well and supplied us with useful data for the
optimization of our project. Moreover, we have made foundational advances in characterising *E. coli* Nissle 1917 and by providing a new application for Cas3 to build a basis for the organism
to be used as live therapeutic. We’re excited to see how scientists will make use of our data
and implement their own probiotics based on our research.

---

[![](https://static.igem.org/mediawiki/2019/1/10/T--Tuebingen--stura_logo.png)](https://www.stura-tuebingen.de/)

[![](https://static.igem.org/mediawiki/2019/9/99/T--Tuebingen--kohlbacher_logo.png)](https://www.kohlbacherlab.org)

[![](https://static.igem.org/mediawiki/2019/8/86/T--Tuebingen--LogoDSS.png)](https://www.dieter-schwarz-stiftung.de/homepage.html)

[![](https://static.igem.org/mediawiki/2019/c/c4/T--Tuebingen--embl_logo.png)](https://www.embl.de/)

[![](https://static.igem.org/mediawiki/2019/e/eb/T--Tuebingen--Logo-Experimenta.png)](https://www.experimenta.science/en/)

[![](https://static.igem.org/mediawiki/2019/e/ec/T--Tuebingen--NCCT.png)](https://www.medizin.uni-tuebingen.de/de/medizinische-fakultaet/forschung/core-facilities/ncct)

---

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