iGEM 2019 — Team IISER_Tirupati

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# Team:IISER Tirupati/Notebook

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Notebook

# Notebook

### May 2019

**1-5-19**
Yeast agglutination assay was done to check for the presence of fimbriae in DH5alpha but no agglutination was observed.

**2-5-19**
Yeast agglutination assay was repeated with modifications but still, no agglutination was observed

**3-5-19**
NEB DH5alpha, NEB BL21, DH5alpha and BL21 were grown on LB plate.

**4-5-19**
Yeast agglutination assay was again repeated following a different protocol, but still no agglutination was observed.

**5-5-19 to 7-5-19**
The different biobricks in the registry were studied and the required ones were selected

**8-5-19**
Yeast was grown in YPD

**9-5-19**
The yeast did not grow in YPD, hence media for growing yeast was prepared manually
Still, no growth of yeast was observed

**10-5-19**
Hemagglutination assay using NEB DH5 alpha and *Pseudomonas* was done but again agglutination was not observed

**11-5-19**
Hemagglutination assay using NEB DH5alpha, BL21 and pseudomonas was repeated following various suggestions from experts but no agglutination was observed

**14-5-19**
Fimbriae were isolated from DH5 alpha and SDS page was done but the bands corresponding to Fimbriae was not observed.
Culture for the preparation of DH5alpha competent cells was set up

**20-5-19**
DH5alpha competent cells were prepared

**19-5-19**
Biobrick was transformed into the competent cells to check the efficiency of the prepared competent cells

**21-5-19**
No colonies were observed after transformation
The protocol for the preparation of competent cell was modified based on various suggestions.
Culture for the preparation of competent cell was set up

**22-05-19**
Competent cells were prepared
Transformation was done to check the efficiency of the prepared competent cells

**23-5-19**
No colonies were observed
Culture for the preparation of ultracompetent cells was setup
Buffers required for the same were prepared

**24-5-19**
Ultracompetent cells were prepared
Transformation was done to check the efficiency of ultracompetent cells
Genomic DNA was isolated

**26-5-19 to 31-5-19**
No colonies appeared
The gblocks and the primers required for making the constructs were designed

### June

**1-6-19**
Agglutination assay using new yeast was done but the results were negative

**10-6-19**
The required biobricks, pSB3k3 plasmid and pSB1C3 plasmid were transformed in NEB DH5alpha competent cells

**13-6-19**
Colonies appeared, so the cultures for the isolation of the plasmids were set up
Lactate assay was done using the lactate detection kit sponsored by Sigma Aldrich
But unexpected results were obtained

**14-6-19**
All the ordered primers arrived
The plasmids were isolated
Genomic DNA was isolated

**14-6-19 to 18-9-19**
lldP, lldR and lldD were amplified from genomic DNA

**20-6-19**
Yeast agglutination assay was repeated using yeast from InStem but again no agglutination was observed

**28-6-19**
BL21 competent cells were prepared

**29-6-19**
pSB1C3 and pSB3K3 plasmids were co-transformed in BL21 and DH5alpha to check the compatibility of plasmids

**29-6-19**
Confirmation tests for co-transformation were performed.

**30-6-19**
Lactate assay was repeated but again the unexpected results were obtained

### July

**1-7-19 to 16-7-19**
HPLC was done to detect the presence of lactate in cancer cells

**17-7-19 to 21-7-19**
Tried to isolate fimbriae using a protocol from literature but it failed again

### August

**1-8-19**
gblocks arrived

**3-8-19**
lldP+lldR was SOEed with prefix-strong promoter-strong RBS+lldP flank and lldR flank-terminator-suffix

**4-8-19**
The above construct was digested using EcoR1 and Pst1 and ligated to digested pSB3K3

**5-8-19**
The ligated plasmids were transformed into DH5alpha

**6-8-19**
Colony PCR was done but positive colonies were not obtained

**7-8-19**
prefix-O1-P-O2-strong RBS-sfGFP flank was SOEed with sfGFP-terminator-suffix
prefix-constitutive promoter- strong RBS-sfGFP flank was SOEed with sfGFP-terminator-suffix

**8-8-19**
The above constructs were digested using EcoR1 and Pst1 and ligated with digested pSB1C3

**9-8-19**
Ligated plasmids were transformed into DH5alpha

**10-8-19**
Colonies appeared and Colony PCR was done
No positive colonies with plasmid containing prefix-O1-P-O2- strong RBS -sfGFP-terminator-suffix were obtained and positive colonies with plasmids containing prefix-constitutive promoter- strong RBS -sfGFP-terminator-suffix were obtained for
Culture for the isolation of positive plasmid was set up

**11-8-9**
The plasmid containing prefix-constitutive promoter- strong RBS -sfGFP-terminator-suffix was isolated
Plasmid linearization and insert release for prefix-constitutive promoter- strong RBS -sfGFP-terminator-suffix was done using prefix and VR2
The released inserts were of appropriate size

**12-8-19**
lldP+lldR was SOEed with prefix-strong promoter-strong RBS-lldP flank and lldR flank-terminator-suffix
The above construct was digested using EcoR1 and PSt1

**13-8-19**
The above construct was ligated to pSB3K3
The ligated plasmid was transformed into DH5alpha

**14-8-19**
Colony PCR was done for the above construct but positive colonies were not obtained
lldP was SOEed with lldD

**15-8-19**
lldP+lldD was SOEed with prefix-strong promoter-strong RBS-lldP flank and lldD flank-terminator-suffix but the end construct after SOEing did not give band of the required length
FimB biobrick was transformed in DH5alpha

**16-8-19**
The culture was set up for plasmid isolation

**17-8-19**
Plasmid containing FimB was isolated
lldD was SOEed with prefix- constitutive strong promoter-strong RBS -lldD flank and lldD flank-terminator-suffix
The end construct did not give band of the required length
The SOEing was repeated but still, the band of required length was not obtained

**18-8-19**
FimB plasmid was transformed into BL21
lldR+lldD was SOEed with prefix-constitutive strong promoter-strong RBS-lldR flank and lldD flank-terminator-suffix, but the end construct obtained was not of the required length

**19-8-19**
DH5alpha and BL21 transformed with FimB plasmid and normal DH5alpha and BL21 were send
to IISER Bhopal for TEM
FimHRPMrel was amplified from the biobrick plasmid.
prefix-O1-P-O2-strong RBS-sfGFP flank was soed with sfGFP-terminator-suffix

**20-8-19**
FimHRPMrel was SOEed with prefix-string promoter-strong RBS-FimHRPMrel flank and FimHRPMrel flank- term-suffix
The prefix-strong promoter-strong RBS -FimHRPMrel-terminator-suffix construct was digested with EcoR1 and Pst1

The prefix-O1-P-O2-strong RBS-sfGFP-terminator-suffix was digested with EcoR1 and Pst1

**21-8-19**
FimHRPMrel construct was ligated with pSB1C3
Transformation of the plasmid containing FimHRPMrel was done
The digested prefix-O1-P-O2 - strong RBS -sfGFP- terminator-suffix construct was ligated with pSB1C3 plasmid
Transformation of the plasmid containing the above construct was done

**22-8-19**
Colony PCR was done for the colonies transformed with prefix-O1-P-O2 - strong RBS-sfGFP - term-suffix and positive colonies were obtained
The 2 constructs of IL12 were SOEed together

**23-8-19**
Plasmid containing prefix-O1-P-O2 -str RBS-sfGFP-term-suffix was isolated from the positive colonies. Linearisation and insert release were done and bands of required length were obtained
Colony PCR was done for the colonies transformed with FimHRPMrel, positive colonies were not observed

**24-8-19**
Colony PCR was again done for the colonies transformed with plasmid containing FimHRPmrel using different set of primers but no positive colonies were observed
IL12 was SOEed with prefix-const strong promoter-strong RBS-IL12 flank and IL12 flank-terminator-suffix, but the construct obtained was not of the required length

**25-8-19**
lldR+lldD was SOEed with prefix-strong promoter-strong RBS -lldR flank and lldD flank-terminator-suffix
The above construct was digested with Ecor1 and Pst1

**26-8-19**
The above construct was ligated with pSB3K3
The ligated product was transformed into DH5alpha

**27-8-19**
colony PCR was done, but again no positive colonies were obtained

**29-8-19**
lldP+lldR was again SOEed with prefix-const strong promoter-strong RBS-lldP flank and lldR flank-terminator-suffix
The above construct was digested with EcoR1 and Pst1

**30-8-19**
The digested construct was ligated with digested pSB3K3 plasmid
The above construct was transformed into DH5alpha

**31-8-19**
Colony PCR was done but no positive colonies were obtained

### September

**1-9-19**
IL12 was soed with prefix-strong promoter-strong RBS-IL12 flank and IL12 flank-terminator-suffix
The above construct was digested with EcoR1 and pst1

**2-9-19**
The SOEed construct was ligated to pSB1C3
The ligated product was transformed into DH5alpha

**3-9-19**
Colony PCR was done and positive colonies were obtained
Culture was setup to isolate plasmid from the positive colonies

**4-9-19**
Plasmids containing IL12 construct was isolated
The isolated plasmid was transformed into BL21

**5-9-19**
prefix-O1-P-O2 -weak RBS-sfGFP flank was SOEed with sfGFp-terminator-suffix
The construct was digested using EcoR1 and pst1

**6-9-19**
The construct was ligated with pSB1C3
The ligated plasmid was transformed into DH5alpha

**7-9-19**
Colony PCR was done and positive colonies were obtained
The culture was setup for plasmid isolation

**8-9-19**
Plasmid containing prefix-O1-P-O2 -weak RBS-sfGFP flank was SOEed with sfGFp-terminator-suffix was isolated.Insert release and linearisation were done and bands of required length were obtained
**9-9-19**
The ordered FimE knockout strain arrived
The FimE knockout strains were plated on LB agar plates

**10-9-19**
Prefix -O1-P O2 -strong RBS-sfGFP flank was SOEed with sfGFP-terminator-suffix
The construct was digested with EcoR1 and Pst1
Culture for the preparation of FimE knockout competent cell was set up

**11-9-19**
The digested construct was ligated with digested pSB1C3 plasmid
The ligated plasmid was transformed into DH5 alpha
FimE knockout competent cells were prepared

**12-9-19**
Colony PCR was done for the colonies transformed with plasmids having Prefix -O1-P-O2-strong RBS-sfGFP-terminator-suffix and positive colonies were obtained
Culture for the isolation of plasmid was set up

**13-9-19**
Plasmid was isolated from the positive colonies.Insert release and linearisation were done and bands of required length were obtained
The plasmid was transformed into BL21

**14-9-19**
FimHRPMrel was SOEed with prefix-strong Promoter-strong RBS-FimHRPMrel flank and fimHRPMrel flank-term-suffix
The construct obtained was digested with EcoR1 and Pst1

**15-9-19**
The digested plasmid was ligated with digested 1C3 plasmid
The plasmid was transformed in DH5alpha

**16-9-19**
Colony PCR was done but positive colonies were not obtained

**17-9-19**
Prefix-constitutive promoter-weak RBS-sfGFP flank was SOEed with sfGFP-terminator-suffix
The SOEed construct was digested with EcoR1 and Pst1

**18-9-19**
The digested construct was ligated with digested pSB1C3 plasmid
The ligated product was transformed into DH5alpha

**19-9-19**
Colony PCR was done and positive colonies were obtained
Culture for the isolation of plasmid was set up

**20-9-19**
The plasmid was isolated.insertrelease and linearisation were done and bands of expected length were obtained
The isolated plasmid and pSB3K3 plasmid were co-transformed into BL21

**21-9-19 to 30-9-19**
Mid semester exams

### October

**1-10-19**
IL12 was SOEed with O1-P-O2-strong promoter-strong RBS-IL12 flank and IL12 flank-terminator-suffix,but the end construct obtained was not of the required size

**2-10-19**
IL12 was again SOEed with O1-P-O2-strong promoter-strong RBS-IL12 flank and Il12 flank-terminator-suffix
The construct obtained was digested with EcoR1 and Pst1

**3-10-19**
The digested construct was ligated with digested pSB1C3
The plasmid was transformed into DH5alpha

**4-10-19**
Colony PCR was done and positive colonies were obtained
Culture for the isolation of the positive plasmids was set up

**5-10-19**
The plasmids were isolated.insert release and linearisation were done and bands of expected length were obtained. The plasmid was transformed into BL21

**6-9-19**
Medium promoter -Strong RBS -lldD -terminator-suffix arrived from twist bioscience
The above construct was digested and ligated with pSB3K3

**7-10-19**
The construct was transformed into DH5alpha
**8-10-19**
The plamids were isolated from the colonies appeared. Insert release and linearisation were done and bands of expected length were obtained
The plasmid was transformed into BL21

**6-10-19 to 17-10-19**
Characterization of the constructs and western blot to detect IL12 was done

**19-10-19**
The lab was cleaned

---

➤

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*"We play with bacteria and sometimes they play us"*

## Contact us

**Email:** igemiisertirupati@gmail.com

**Address:**
 Indian Institute of Science Education and Research, Tirupati
C/o Sree Rama Engineering College (Transit Campus),
Rami Reddy Nagar, Karakambadi Road,
Mangalam (P.O.) Tirupati -517507.
Andhra Pradesh, INDIA

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# Team:IISER Tirupati/Experiments

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Wet Lab

[Experiments](https://2019.igem.org/Team%3AIISER_Tirupati/Experiments)
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[Notebook](https://2019.igem.org/Team%3AIISER_Tirupati/Notebook)
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[Mathematical Model](https://2019.igem.org/Team%3AIISER_Tirupati/Model)
[Website](https://2019.igem.org/Team%3AIISER_Tirupati/Website)

Team

[Members](https://2019.igem.org/Team%3AIISER_Tirupati/Team)
[Collaborations](https://2019.igem.org/Team%3AIISER_Tirupati/Collaborations)

Human Practice

[Integrated Human Practices](https://2019.igem.org/Team%3AIISER_Tirupati/Human_Practices)
[Outreach](https://2019.igem.org/Team%3AIISER_Tirupati/Public_Engagement)

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[Achievements](https://2019.igem.org/Team%3AIISER_Tirupati/Achievements)

Experiments

![exp](https://static.igem.org/mediawiki/2019/8/8b/T--IISER_Tirupati--exp-cov.jpeg)

EXPERIMENTS

* [Cloning](#tab1)
* [HPLC](#tab2)
* [Characterization](#tab3)

We used DH5a strain of *E. Coli* for cloning and BL21 (DE3) as chassis/expression vector. sfGFP was used as reporters in our constructs. The plasmids we used were psb1C3 (promoter regions and sfGFP) and psb3K3 (structural elements of lldPRD operon).

The fragments of choice were ordered and were ligated together using SOEing wherever necessary, and the clones were confirmed by colony PCR and insert release. We chose SOEing for our constructs because it provided us with a cost effective option that could give us our library of constructs within a short span of time.

Simultaneously, our constructs were sent for sequencing as we got them.

For our constructs, we chose to use RBS of different strengths, since a library of various promoters was already screened (ETH Zurich). From that library, we chose the best promoter - J23117. The following constructs from our library were successfully cloned:

1. Constitutive Promoter - Strong RBS - sfGFP - Terminator- O1 - J23117 - O2 - Strong RBS - sfGFP - Terminator- Constitutive Promoter - Weak RBS - sfGFP - Terminator- O1 - J23117 - O2 - Weak RBS - sfGFP - Terminator- Constitutive Promoter - Strong RBS - YebF-IL12-Flag-Terminator- O1 - J23117 - O2 - Strong RBS - YebF-IL12-Flag- Terminator- Medium Promoter - Strong RBS - lldD - Terminator

 [Protocols](https://2019.igem.org/Team%3AIISER_Tirupati/Protocols#tab1) [Results](https://2019.igem.org/Team%3AIISER_Tirupati/Results#tab1)

### Theory

There are many characteristic properties of cancer cells which differentiate them from healthy cells. One among them is the production of lactic acid and thereby, a lactate rich microenvironment [1]. We use this property to helps our CoCa coli to gain specificity in its function.
Cancer cells reprogramme their metabolism to promote growth, proliferation, and survival. One of the methods is by uptaking more glucose. In cancer cells, glucose uptake increases and fermentation of glucose to lactate takes place even in the presence of completely functioning mitochondria and oxygen. This phenomenon is known as the Warburg effect [2].
The amount of respiration and lactate production varies depending on genetic or environmental influences [3]; thus, it will be different for different tumours. Even though aerobic respiration produces less number of ATP compared to mitochondrial respiration, the rate of glucose metabolism is more in that. This higher rate of metabolism and lower production of ATP is an evolutionary adaptation of cancer cells, which gives them an advantage over healthy cells for limited glucose and nutrients [4].
Warburg effect also supports the biosynthetic requirements of cancer cells, helping them with the uncontrolled proliferation [5].
The increased glucose consumption decreases the pH of tumour microenvironment due to lactate secretion, and this helps the cancer cells to spread quickly [6]. Also, the high rate of glycolysis helps the cancer cells to get more glucose than the Tumour Infiltrating Lymphocytes (TIL) and thereby to escape from the body’s immune system [7]. Thus, the Warburg Effect provides an overall benefit that supports tumour microenvironment to help in cell proliferation and survival.

### Detection and quantification

Knowing the amount of lactic acid produced by the cancerous and healthy cells would help us tune our constructs. To detect the amount of lactate produced by the cancer cells, we decided to use HPLC (High-Performance Liquid Chromatography).
Since our project is about colon cancer, we decided to use HT-29 (colon cancer cell line) for the detection and quantification of lactate present in the tumour microenvironment, and CRL-1790 (normal colon cell line) was used as a negative control.

 [Protocols](https://2019.igem.org/Team%3AIISER_Tirupati/Protocols#tab2) [Results](https://2019.igem.org/Team%3AIISER_Tirupati/Results#tab2)

---

**References:**

1. Geoffrey M Cooper. - The cell, a molecular approach; Development and causes of cancer- Maria V. Liberti and Jason W. Locasale (2016). The Warburg Effect: How Does it Benefit Cancer Cells?- Crabtree HG. Observations on the carbohydrate metabolism of tumours. Biochemical journal. 1929;23(3):536- Pfeiffer T, Schuster S, Bonhoeffer S. Cooperation and competition in the evolution of ATP-producing pathways. Science. 2001;292(5516):504–507.- Vander Heiden MG, Cantley LC, Thompson CB. Understanding the Warburg effect: the metabolic requirements of cell proliferation. science. 2009;324(5930):1029–1033.- Estrella V, et al. Acidity generated by the tumor microenvironment drives local invasion. Cancer research. 2013;73(5):1524–1535.- Chang C-H, et al. Metabolic Competition in the Tumor Microenvironment Is a Driver of Cancer Progression. Cell. 2015

For our final step, we characterised each of our sfGFP constructs after induction with several different concentrations of lactate. The lactate inducible YebF-IL12 construct was also characterised using western blotting. Throughout our assay, we used sfGFP as our reporter instead of IL12 because of the easily quantifiable fluorescence read-out along with its increased in vitro thermal stability, improved folding kinetics and superior resistance against chemical denaturants in comparison to conventional GFPs.

Our constructs have two kinds of regulatory sequences:

1. **O1PO2:** To make our bacteria sensitive to the lactate that is secreted by colon cancer cells, we used this region from the bacterial lactate operon. Along with this, different types of RBS were used to modulate the sensitivity of the operon to varying levels of lactate in the colon cancer microenvironment.- **Constitutive promoter:** This promoter was used to measure the levels of our reporter protein in the constructs. These levels then served as controls with which we compared the production levels of O1PO2. Hence, we could determine the difference between the two and check if our fine tuning was working.

The detailed protocol is available on the protocols page. Check out our results page too!

 [Protocols](https://2019.igem.org/Team%3AIISER_Tirupati/Protocols#tab3) [Results](https://2019.igem.org/Team%3AIISER_Tirupati/Results#tab3)

---

**References:**
Goers, L. , Ainsworth, C. , Goey, C. H., Kontoravdi, C. , Freemont, P. S. and Polizzi, K. M. (2017), Whole‐cell Escherichia coli lactate biosensor for monitoring mammalian cell cultures during biopharmaceutical production. Biotechnol. Bioeng., 114: 1290-1300. doi:10.1002/bit.26254

➤

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*"We play with bacteria and sometimes they play us"*

## Contact us

**Email:** igemiisertirupati@gmail.com

**Address:**
 Indian Institute of Science Education and Research, Tirupati
C/o Sree Rama Engineering College (Transit Campus),
Rami Reddy Nagar, Karakambadi Road,
Mangalam (P.O.) Tirupati -517507.
Andhra Pradesh, INDIA

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## Our sponsors

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# Team:IISER Tirupati/Results

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Project

[Inspiration](https://2019.igem.org/Team%3AIISER_Tirupati/Inspiration)
[Description](https://2019.igem.org/Team%3AIISER_Tirupati/Description)
[Design](https://2019.igem.org/Team%3AIISER_Tirupati/Design)
[Results](https://2019.igem.org/Team%3AIISER_Tirupati/Results)
[Contribution](https://2019.igem.org/Team%3AIISER_Tirupati/Contribution)
[Improve](https://2019.igem.org/Team%3AIISER_Tirupati/Improve)
[Attributions](https://2019.igem.org/Team%3AIISER_Tirupati/Attributions)

Wet Lab

[Experiments](https://2019.igem.org/Team%3AIISER_Tirupati/Experiments)
[Protocols](https://2019.igem.org/Team%3AIISER_Tirupati/Protocols)
[Notebook](https://2019.igem.org/Team%3AIISER_Tirupati/Notebook)
[Parts](https://2019.igem.org/Team%3AIISER_Tirupati/Parts)
[Safety](https://2019.igem.org/Team%3AIISER_Tirupati/Safety)

Dry Lab

[Mathematical Model](https://2019.igem.org/Team%3AIISER_Tirupati/Model)
[Website](https://2019.igem.org/Team%3AIISER_Tirupati/Website)

Team

[Members](https://2019.igem.org/Team%3AIISER_Tirupati/Team)
[Collaborations](https://2019.igem.org/Team%3AIISER_Tirupati/Collaborations)

Human Practice

[Integrated Human Practices](https://2019.igem.org/Team%3AIISER_Tirupati/Human_Practices)
[Outreach](https://2019.igem.org/Team%3AIISER_Tirupati/Public_Engagement)

☰
[Achievements](https://2019.igem.org/Team%3AIISER_Tirupati/Achievements)

Experiments

![exp](https://static.igem.org/mediawiki/2019/b/bc/T--IISER_Tirupati--rescov.jpeg)

RESULTS

* [Cloning](#tab1)
* [HPLC](#tab2)
* [Characterization](#tab3)

## Construct 1: O1PO2-Strong RBS with sfGFP-terminator [(BBa\_K3110040)](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3110040)

#### SOEing of O1PO2-Strong RBS with sfGFP-terminator

The following gel image shows the band obtained after SOEing O1PO2-Strong RBS with sfGFP-terminator. As expected, a band of 1061 bp was obtained.

![](https://static.igem.org/mediawiki/2019/d/d9/T--IISER_Tirupati--G1.png)

The obtained construct was then ligated into pSB1C3 plasmid and transformed into DH5alpha.

#### Selection of positive colonies

Colony PCR was done to select the positive colonies.
The following gel image shows the band obtained after performing colony PCR with Prefix and VR primers. 1 colony out of 4 gave band at 1.2 kb as expected.

![](https://static.igem.org/mediawiki/2019/c/c8/T--IISER_Tirupati--G2.png)

#### Confirmation of the presence of correct plasmids in the selected colonies

Plasmids from the positive colonies were isolated. Linearisation of the plasmid and insert release using the appropriate restriction enzyme were done to confirm that the selected colonies are positive.
The following gel image shows the bands obtained on linearisation and insert release. As expected, a band of 3.1 kb was obtained on linearisation and 2 bands of 1061 bp and 2.1 kb was obtained upon insert release.

![](https://static.igem.org/mediawiki/2019/c/c5/T--IISER_Tirupati--G3.png)

## Construct 2: Constitutive P-Strong RBS-sfGFP-terminator [(BBa\_K3110043)](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3110043)

#### SOEing of Constitutive P-Strong RBS with sfGFP-terminator

![](https://static.igem.org/mediawiki/2019/e/ec/T--IISER_Tirupati--G4.png)

The obtained construct was ligated into pSB1C3 plasmid and transformed in DH5alpha.

#### Selection of positive colonies

Positive colonies were selected by performing colony PCR using prefix and VR primers. 4 colonies out of 5 gave band at 1.1 kb as expected.

![](https://static.igem.org/mediawiki/2019/6/64/T--IISER_Tirupati--G5.png)

#### Confirmation of the presence of correct plasmids in the selected colonies

As expected, a band of 3 kb was obtained on linearisation and 2 bands of 950 bp and 2.1 kb was obtained on insert release.

![](https://static.igem.org/mediawiki/2019/b/b9/T--IISER_Tirupati--G6.png)

## Construct 3: O1PO2-Weak RBS-sfGFP-Terminator [(BBa\_K3110042)](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3110042)

#### SOEing of O1PO2-Weak RBS with sfGFP-Terminator

This construct was designed to give us a weaker expression of sfGFP as compared to the previous constructs. O1PO2-Weak RBS was SOEed with sfGFP-Terminator and as expected a band of 1060 bp was obtained.

![](https://static.igem.org/mediawiki/2019/4/4f/T--IISER_Tirupati--G7.png)

The obtained construct was ligated into pSB1C3 plasmid and transformed in DH5alpha.

#### Selection of positive colonies

Colony PCR was done to select the positive colonies
The following gel image shows the band obtained after performing colony PCR with prefix and VR primers. 10 colonies out of 10 gave band at 1.2 kb as expected.

![](https://static.igem.org/mediawiki/2019/7/78/T--IISER_Tirupati--G8.png)

#### Confirmation of the presence of correct plasmids in the selected colonies

As expected, a band of 3.1 kb was obtained on linearisation and 2 band of 1060 bp and 2.1 kb was obtained on insert release.

![](https://static.igem.org/mediawiki/2019/9/9c/T--IISER_Tirupati--G9.png)

## Construct 4: Constitutive P-Weak RBS-sfGFP-Terminator [(BBa\_K3110045)](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3110045)

#### SOEing of Constitutive P-Weak RBS with sfGFP-Terminator

This construct was designed as a control for construct 3.The following gel image shows the band obtained after SOEing Constitutive P-Strong RBS with sfGFP-Terminator. As expected, a band of 950 bp was obtained.

![](https://static.igem.org/mediawiki/2019/c/c6/T--IISER_Tirupati--G10.png)

The obtained construct was ligated into pSB1C3 plasmid and transformed in DH5alpha

#### Selection of positive colonies

Colony PCR was done to select the positive colonies
The following gel image shows the band obtained after performing colony PCR with prefix and VR primers. 9 colonies out of 9 gave band at 1.1 kb as expected.

![](https://static.igem.org/mediawiki/2019/9/93/T--IISER_Tirupati--G11.png)

#### Confirmation of the presence of correct plasmids in the selected colonies

Plasmids from the positive colonies were isolated. As expected, a band of 3 kb was obtained on linearisation and 2 band of 950 bp and 2.1 kb was obtained on insert release.

![](https://static.igem.org/mediawiki/2019/4/44/T--IISER_Tirupati--G12.png)

## Construct 5: O1PO2-Strong RBS -YebF IL12 FLAG-Terminator [(BBa\_K3110051)](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3110051)

#### SOEing of O1PO2-Strong RBS -YebF IL12 FLAG-Terminator

This construct was a part of our IL12 secretory system. While positive clones were obtained, IL12 wasn’t detected either in the cell pellet or in the supernatant. We hypothesise a faulty SOEing that could’ve caused a errors in our promoter or RBS regions.
The following gel image shows the band obtained after SOEing O1PO2-Strong RBS with -YebF IL12 FLAG-Terminator. As expected, a band of 2310 bp was obtained.

![](https://static.igem.org/mediawiki/2019/c/cf/T--IISER_Tirupati--G13.png)

The obtained construct was ligated to pSB1C3 plasmid and transformed in DH5alpha.

#### Selection of positive colonies

Colony PCR was done to select the positive colonies.
The following gel image shows the band obtained after performing colony PCR with VF2 and VR primers. 5 out of 9 colonies gave band at 2.5 kb as expected.

![](https://static.igem.org/mediawiki/2019/7/74/T--IISER_Tirupati--G14.png)

#### Confirmation of the presence of correct plasmids in the selected colonies

The following gel image shows the bands obtained on linearisation and insert release. As expected, a band of 4.4 kb was obtained on linearisation and 2 band of 2.3 kb and 2.1 kb were obtained on insert release.

![](https://static.igem.org/mediawiki/2019/9/91/T--IISER_Tirupati--G15.png)

## Construct 6: Constitutive P-Strong RBS -YebF IL12 FLAG-Terminator [(BBa\_K3110054)](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3110054)

#### SOEing of Const Strong P-Strong RBS -YebF IL12 FLAG-Terminator

This construct was designed as a control for the construct 5, and was supposed to show higher protein expression. The following gel image shows the band obtained after SOEing Const. Strong P-Strong RBS with -YebF IL12-Terminator. As expected, a band of 2205 bp was obtained.

![](https://static.igem.org/mediawiki/2019/9/9b/T--IISER_Tirupati--G16.png)

The obtained construct was ligated to psB1C3 plasmid and transformed in DH5alpha.

#### Selection of positive colonies

Colony PCR was done to select the positive colonies
The following gel image shows the band obtained after performing colony PCR with VF2 and VR primers. 4 colonies out of 10 gave band at 2.5 kb as expected.

![](https://static.igem.org/mediawiki/2019/8/88/T--IISER_Tirupati--G17.png)

#### Confirmation of the presence of correct plasmids in the selected colonies

Linearisation of the plasmid and insert release using the corresponding primers were done to confirm that the selected colonies are positive.
The following gel image shows the bands obtained on linearisation and insert release. As expected, a band of 4.3 kb was obtained on linearisation and 2 band of 2.2 kb and 2.1 kb was obtained on insert release.

![](https://static.igem.org/mediawiki/2019/c/c2/T--IISER_Tirupati--G18.png)

## Construct 7: Medium P - Strong RBS - lldD -Terminator [(BBa\_K3110007)](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3110007)

#### Confirmation of the presence of correct plasmids in the selected colonies

The construct was received from twist bioscience was ligated to pSB3K3 and transformed in DH5alpha.The following gel image shows the bands obtained on linearisation and insert release. As expected, a band of 4.2 kb was obtained on linearisation and 2 band of 1500 bp and 2.7 kb was obtained on insert release.

![](https://static.igem.org/mediawiki/2019/f/f2/T--IISER_Tirupati--G19.png)

## Construct 8: Constitutive P - Strong RBS - FimHRPMrel -Terminator [(BBa\_K3110047)](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3110047)

#### SOEing of Constitutive P - Strong RBS - FimHRPMrel -Terminator

This construct was made to help our bacteria secrete our colon cancer homing peptide at the tip of its fimbriae. The constitutive strong promoter used here is J23100.
The following gel image shows the band obtained after SOEing Constitutive P-Strong RBS with FimHRPMrel -Terminator. As expected, a band of 1250 bp was obtained.

![](https://static.igem.org/mediawiki/2019/b/b1/T--IISER_Tirupati--G20.png)

The obtained construct was ligated to pSB1C3 plasmid and transformed in DH5alpha.

#### Selection of positive colonies

Colony PCR was done to select the positive colonies.
The following gel image shows the band obtained after performing colony PCR with VF2 and VR primers. No colonies gave band at the expected size of 1.4 kb.

### Overlaid chromatogram of standard solutions.

This graph tells us about the retention time of L-lactate in the mobile phase which was used. As two peaks were observed, we collected the elute corresponding to the two retention times and performed a mass spectrometry (data not shown) to confirm which is the L-lactate peak. **The peak at retention time 3.9 minutes corresponded to lactate.** It is important to note that the overlaid graphs are increasing concentrations of lactate solution (the ones we prepared for standard curve plotting) and not spiking.

![](https://static.igem.org/mediawiki/2019/6/6c/T--IISER_Tirupati--H2.png)

### Standard Graph with Error Bar

This standard curve of L-lactate plotted using the areas under the peak of 5 different concentrations helps us find out the concentration of HT-29 cell’s spent media. We obtained the value from literature to be ~18 mM and hence we chose concentrations for the curve ranging from 10 mM to 100 mM.

![](https://static.igem.org/mediawiki/2019/9/96/T--IISER_Tirupati--H1.png)

### Overlaid chromatogram of spent media and spiking (24 hours-HT-29)

This graph is obtained by injecting the spent media of a 24-hour culture of HT-29 grown in a 60 mm dish. A peak is obtained with a similar retention time of that of the standard L-lactate solutions, which indicates that the media has L-lactate in it. To confirm that the peak is solely due to L-lactate, we injected increasing concentrations of L-lactate along with the media (spiking) and a corresponding increase of area under the peak was observed. Thus, we can conclude that the peak obtained from spent media is that of L-lactate.
Biological triplicates of this experiment yielded lactate peaks with different areas which when interpolated on the standard curve gave us an average value of **16 mM** lactate.

![](https://static.igem.org/mediawiki/2019/6/6d/T--IISER_Tirupati--H3.png)

### Overlaid chromatogram of RPMI media and spent media (24 hours)

The purple line is the curve obtained by injecting fresh RPMI media with 10% FBS.No significant peak was observed at lactate’s retention time (3.9 mins). The overlaid black line is the readout from spent media showing a visible lactate peak.

![](https://static.igem.org/mediawiki/2019/6/6b/T--IISER_Tirupati--H4.png)

### Characterising the sfGFP Constructs

The regulatory element lldR binds to lldRO1 and lldRO2 (operator regions of lldPRD operon) and inhibits transcription. J23117 is a promoter intercalated between the operators. lldR dimer represses the transcription possibly by forming a DNA loop which doesn’t allow the RNA polymerase to bind the promoter. Upon binding of L-Lactate to lldR, however, this transcriptional suppression is lost and instead lldR complex with L-Lactate remains bound to LldRO1 acting as a transcriptional activator.

lldRO1-J23117-lldRO2 was **characterized using sfGFP** as the reporter gene under the control of RBS of different strengths, namely, Strong, Medium and Weak(Part:BBa\_K3110040, Part:BBa\_K3110041, Part:BBa\_K3110042).

To test this part we have designed two plasmids:
O1PO2- RBS-sfGFP-terminator in a medium copy plasmid pSB1C3
P-RBS-sfGFP-terminator in another pSB1C3 (Same Promoter without the operator regions) - as a control

On adding lactate to the culture, L-lactate binds to lldR thus leading to enhanced expression of sfGFP only when O1PO2 is upstream of sfGFP.

![](https://static.igem.org/mediawiki/2019/2/2a/T--IISER_Tirupati--strfluo.jpeg)

![](https://static.igem.org/mediawiki/2019/2/27/T--IISER_Tirupati--c3.jpeg)

Graph 1

As can be seen in the **graph 1**, when P-strongRBS-sfGFP is there, the fluorescence level is not varying much with an increase in L-lactate concentration. But, in the case of O1PO2-strongRBS-sfGFP, the increase in lactate concentration has lead to an increase in the fluorescence output.

![](https://static.igem.org/mediawiki/2019/5/58/T--IISER_Tirupati--weakfluor.jpeg)

![](https://static.igem.org/mediawiki/2019/7/7b/T--IISER_Tirupati--c4.png)

Graph 2

In the case of weak RBS however, a clear increase wasn’t observed **(Graph 2)** and hence strong RBS is an optimum construct to use.
As the lactate concentration is increasing, the amount of free lldR (L-Lactate Repressor) inside the cell is decreasing as well as the lldR bound to lactate (activator) is increasing - so, the rate of sfGFP expression is also increasing with increasing Lactate (up to a certain concentration). After lldR is saturated with L-lactate, the sfGFP expression becomes steady. The lactate concentration of around 20 mM has however provided an unexpected decrease in normalized Fluorescence and further experimentation is required to validate the reasons for the same.
The construct with strong RBS gives a clear **sigmoidal** pattern for detection of lactate with a **threshold of around 0.45 mM.** This threshold doesn't help us since the construct will fail to distinguish Cancerous vs Non-cancerous microenvironments, both of which have lactate concentration higher than 0.45 mM.

Hence we have intended to use this construct in combination with our library of four gene constructs: lldD, lldP+lldD, lldR+lldD and lldP+lldR+lldD under promoter and RBS of different strengths.

![](https://static.igem.org/mediawiki/2019/7/75/T--IISER_Tirupati--c2.png)

Graph 3

We have characterized the **new part Medium Promoter Strong RBS lldD** which was cloned into pSB3K3 (a low-copy number plasmid) which is compatible with pSB1C3. This plasmid along with lldRO1-J23117-lldRO2 Strong RBS sfGFP (which was cloned into pSB3K3) was co-transformed into BL21 (DE3) and the assay was carried out. lldD (L-Lactate Dehydrogenase) cleaves L-lactate to Pyruvate thus depleting the amount of lactate in the cell.
This, we thought, would be the best way to control the expression threshold of lldRO1-J23117-lldRO2 **(Graph 3)**.

![](https://static.igem.org/mediawiki/2019/8/85/T--IISER_Tirupati--c1.jpeg)

Graph 4

Ectopic expression of lldD should lead to lower levels of Lactate in the cell which in turn would lead to higher levels of free lldR for transcription inactivation. And a higher dose of lactate would be required to turn on the expression. And indeed this is what we observed **(Graph 4)**. We observed an increase in threshold from 0.45 mM (without lldD) to 2 mM (with lldD). Although this won’t be able to differentiate cancer from non-cancerous cells, the use of a strong promoter upstream to lldD and an appropriate combination of lldR and lldP in addition to lldD should. However, ***an increase of threshold suggests that our idea of using lldD as the modulator will work.*** A major drawback that we observed was a decrease in sensitivity i.e. even at sub-threshold lactate levels, the fluorescence observed in the presence of lldD was high. Not only this, at the aforementioned threshold levels, the intensity was lower than in the case without lldD. This could be rectified by using lldP and lldR along with lldD.

---

### YebF IL12 FLAG Secretion

For both the YebF IL12 FLAG constructs, we wanted to see if the protein was being produced. To check for protein production, we cultured BL21 (DE3) transformed with the plasmids and collected both the pellet and supernatant of the overnight culture. For the constitutive promoter construct, the control was a culture of non-transformed BL21 (WT).In case of the lactate inducible promoter construct, a **1 mM lactate induction** was given and this induced culture was compared to an uninduced one.

The gel images below show **no visible band difference** between the pellet and supernatant fractions of the samples. This indicates that the protein itself was not produced.

![](https://static.igem.org/mediawiki/2019/d/d0/T--IISER_Tirupati--constP1l12.jpeg)

![](https://static.igem.org/mediawiki/2019/f/fe/T--IISER_Tirupati--o1po2il12.jpeg)

In order to verify the production of IL12, we performed a western blot with the inducible O1PO2-Strong RBS -YebF IL12 FLAG-Terminator construct. We induced BL21 carrying this construct with 1 mM lactate and had an uninduced control to compare to. Since our protein (**74.04 kDa**) has a FLAG tag, we probed the membrane with an anti-FLAG antibody (raised in mouse) and then imaged using a secondary anti-mouse HrP conjugated antibody. We have also loaded a positive control to ensure that the antibody worked fine.

The blot image shown below was obtained after a **10 minute** exposure. The positive control shows the expected size (~36 kDa) while our samples show some non-specific bands (~35kDa).

![](https://static.igem.org/mediawiki/2019/d/d2/T--IISER_Tirupati--western.jpeg)

We thus concluded that the bacteria **did not produce** the YebF IL12 FLAG protein. We suspected that this is happening due to SOEing errors. Indeed, we confirmed this possibility after obtaining the sequencing results. However, we could not repeat the experiment with a new clone due to time constraints.

➤