Updated on 2021-11-18: You might found this file, which we prepared for the Judging Session, helpful to understand our goals, approaches and what have been achieved.
# Detection of C. albicans through LAMP and LFA
# Testing the availability of primers and the DNA in LAMP
First, to affirm the system could work properly, we started from the very beginning by testing the availability of our primers and the DNA extracted from Candida albicans by our instructor following safety rules. As Figure 1 indicates, we got positive results with both agarose gel electrophoresis and LFA.
Figure 1. positive and specific test of LAMP combined with LFA. M: DL2000 marker; N:negative control (replacing DNA
of C. albicans with *S. cerevisia); C:*positive amplification of the DNA of C. alibicans
# Identifying reaction conditions
Next, we tried to define the conditions to carry out the detection, including the detection limit, the minimum amount of enzyme required in the reaction, reaction time, and temperature. The reaction time available is above 70 mins. The reaction temperature ranges from 60-65 °C. The minimum amount of enzyme catalyzing LAMP is 2 U/25μl, and the detection limit of C. albicans using LAMP is 0.4 pg/μl. 0.4 pg/μl.
Figure 2. Testing detection conditions. (a) Testing reaction time. M: marker; Other lanes: different reaction time as indicated.
(b) Testing minimum enzyme requirement to react. M: DL2000 marker; other lanes: different reaction temperature as indicated. (c) Testing reaction temperature. M: DL2000 marker, N: negative
control; other lanes: the different amount of enzyme as indicated. (d) The testing detection limit of LAMP combined with LFA. M: DL2000 marker; other lanes: different template concentration as indicated.
# Functions of T7 gene products
In our project, the T7 gene product circuit is used to inhibit the endogenous transcription of E. coli, thus enhancing the purity of the protein of interest. To test T7 gp2 and gp5.7, which are respectively known as σ70 inhibitor and σS inhibitor, we must first construct σS promoters for following testing.
σS promoters verification
Six σS promoters core region sequences are tested using growth curve measurement and flourescence intensity curve measurement protocol.
All of the six sequences are proved to be σS promoters that can function independently. Among them, pdps shows the highest strength as a σS promoter.
Further results can be viewd at the registry page.
http://parts.igem.org/Part:BBa_K3790014
Functions of T7 gene product 2
All the relevant results can be viewd at the registry page.
Please visit:
http://parts.igem.org/Part:BBa_K3790231
Functions of T7 gene product 2
All the relevant results can be viewd at the registry page.
Please visit:
http://parts.igem.org/Part:BBa_K3790232
# T7 driven Bst expression
In our project, the T7 expression system (based on the pET52 plasmid) was used to express the large fragment of Bst DNA polymerase I (large fragment), which is required for the isothermal amplification reaction (LAMP to be specific).
Our Bst (or previously called BstPol) is derived from the DNA polymerase I large fragment in Bacillus stearothermophilus. The large fragment of Bst DNA Polymerase I contains 5' to 3' DNA polymerase activity and strong strand displacement activity, but lacks 5' to 3' exonuclease activity, ideal for target sequence amplification.
# Construction of K3700212
We obtained the Bst polymerase large fragment coding DNA optimized for Escherichia coli codon from Twist Bioscience, by total synthesis. We have the pET backbone in our laboratory stocks (namely pET16, pET28 and pET52; all of three shared the same backbone sequence used in our experiments).
We used ClonExpress® Homologous Recombination Kit from Vazyme to perform PCR based cloning, placing Bst (Figure 1) between T7 RBS and T7 terminator. Homology arm primers used are documented at the Design page.
Figure 1. Gel electrophoresis of PCR produts, using Twist synthesis as the template. The first lane was loaded with DNA ladder,
sizes were marked on the image. The brightest band of 750 bp was about 100 ng, and other bands about 50 ng. The arrowhead marks the target DNA band, approximately 1800bp.
The Bst DNA polymerase I large fragment coding DNA is sized at 1764bp, which is approximately 1800bp after adding homology arms to both ends by PCR reaction. The result (Figure 1) shows a successful amplification of the DNA of interest. We performed gel extraction of the target region, to obtain pure Bst with homology arms for subsequent reactions.
Figure 2. Gel electrophoresis of linearized pET bone. The first lane was loaded with DNA ladder, sizes were marked
on the image. All bands are about 50 ng. The pET plasmid was subjected to loop PCR reaction with homology arm primers in order to linearize the vector. Clone16 and Clone52 were amplified from two different pET stock
DNA templates. The arrowhead marks the target approximately 5200 bp.
Figure 2 indicates that we have obtained two copies of correct sized linearized pET backbone.
After ClonExpress® homologous recombination reaction, Fast-T1 competent bacteria were transformed with reactants, and spread onto LB plates with ampicillin. We picked colony after 16 h culturing plates at 37℃. We further grow clones in 2 ml LB liquid medium with ampicillin, 37℃ shaking overnight.
Mini-prep was performed the following day, and the resulting plasmids were sent to Sanger sequencing (one correct sequencing result is shown in Figure 3).
Figure 3. Screenshot of one correct sequencing result, only small portion is shown. This clone was obtained using Clone52 linearized
pET backbone. We sequenced the plasmids from both ends of Bst, and the valid sequences overlapped.
The plasmids containing sequencing verified K3790212 were restriction digested with NcoI and KpnI. The correct digestion pattern is shown in Figure 4.
Figure 4. Gel electrophoresis of plasmids containing K3790212 digested with NcoI and KpnI. The first lane was loaded with DNA
ladder, sizes were marked on the image. All bands are about 50 ng. Lane labeled with K3790212 is the plasmid DNA without enzymatic digestion. Lane labeled with NcoI/KpnI, is digested. The arrowhead marks the predicted
excised DNA fragment approximately 1800bp.
# Bst Expression
The correct BL21 (DE3) colonies were picked into 2 ml ampicillin LB liquid medium overnight with shaking at 37 ℃.
The following day, the culture was transferred to 2 ml ampicillin LB broth at a 2% volume ratio, and incubation continued at 37 ℃ until reach the logarithmic growth phase of the bacteria (OD600 ~ 0.6). We then started IPTG induction, for at least 3 hours.
Figure 5. Gel electrophoresis of colony PCR of BL21 (DE3) clones. The first lane was loaded with DNA ladder, sizes were marked
on the image. The brightest band of 750 bp was about 100 ng, and other bands about 50 ng. After obtaining the correct K3790212 recombinant plasmids, we transformed them into BL21 (DE3) competent bacteria for protein
expression. Positive colonies were selected by ampicillin preliminarily and then verified by colony PCR. The arrowhead marks the target approximately 1800bp.
The induced bacterial culture were measure (OD600 absorbance). Some of the bacterial culture was centrifuged to collect a pellet, and a self-made Bst polymerase compatible buffer (BPCB) was added (for formula, see Table 1). After lysis and 4 ℃ 13000rpm 10-min centrifugation, we collected the supernatant (containing Bst polymerase, Figure 6), to perform LAMP reaction. Another portion of the bacterial culture was directly centrifuged and the pellet was resuspended in 1x SDS sample buffer followed by immediate boiling at 100 ℃ for 5 minutes, will be used for protein SDS-PAGE (Figure 8).
Figure 6. Compare proteins in the pellet vs. supernatant, after BPCB lysis.Lane 2 was Bst containing supernant.
We add equal volume of 2x SDS sample buffer. Lane 1 was BPCB insolvable components, from equivalent pellet. As shown in the gel, BPCB solubilizes the bacteria very well, and we do not perform 4 ℃ centrifugation after
seeing this gel.
# Bst in BPCB for LAMP
The two lanes in Figure 7 show that Candida albicans DNA strongly amplified by a lysate of K3790212 containing, IPTG induced, BL21 (DE3) bacteria. The specificity of DNA product was later verified with lateral flow assay (LFA).
Figure 7. LAMP reactions using purified Bst or self-made bacterial lysates. The first lane was loaded with DNA ladder, sizes
were marked on the image. The brightest band of 750 bp was about 100 ng, and other bands about 50 ng. For the last two lanes, we used bacterial lysates from BL21 (DE3) transformed with K37900212, with or without IPTG
induction. Purified Bst represents the purified enzyme.
# The initial test with our gp5.7 plasmid
Figure 8. Compare whole bacteria protein under different induction condition.BL21 (DE3) bacteria, transformed
with either K3790212 alone, or with both K3790212 and K3790232, were cultured to OD600 ~ 0.6 and then induced as indicated for 3 hours. Whole cell lysates were prepared by directly adding 1x SDS sample buffer into bacteria
pellets, followed by immediate boiling at 100 ℃ for 5 minutes. Lane 5 and Figure 6, was made using a same culture, IPTG induced for 3 hours.
# Fusion protein acquisition
In our project, we wanted to make certain modifications to the DNA polymerase of the LAMP reactionto improve its relevant activity and thus the detection limit and precision of the test strips we designed. In the course of previous studies, we found that the addition of sso7d double-stranded binding protein tended to increase the activity of DNA polymerase, but there is a lack of relevant literature on sso7d to increase the activity of Bst Pol. Therefore we ventured to hypothesize that sso7d could enhance Bst Pol activity, and we selected three similar double-stranded binding proteins from the same species as sso7d, Sulfolobus solfataricus. Also, to verify whether single-chain binding proteins also have similar effects, two single-chain binding proteins from Sulfolobus solfataricus and E. coli, respectively, were selected for the experiment. We also considered that different binding sites of the binding proteins to the polymerase might lead to different results, and therefore we designed whether the binding proteins are attached to the C- or N-terminal end of Bst Pol, respectively.
To construct the fusion proteins, we need to obtain the DNA of Bst Pol and the six binding proteins and ligate Bst Pol and the six binding proteins in the designed positions by homologous recombination.
# Acquisition of six binding protein DNAs
We have obtained fully synthesized Bst Pol DNA from Twist, and we chose to synthesize the binding proteins by oligo assembly by PCR because of the small number of bases in the six binding proteins.
Figure 9. Assembled DNA binding proteins, albA1, S.ssb, E.ssb. The first lane was loaded with a DNA ladder, sizes were marked on the image.
The brightest band of 750 bp was about 100 ng and other bands about 50 ng. Lanes with correct sized amplified DNA were labeled. After PCR cloning, several bacterial clones were picked, grew into cultures, and sent for
Sanger sequencing. Then, we verified the sequencing results and used the correct ones for further experiments.
Figure 10. Assembled DNA binding proteins, DbpA, sso10b, sso7d. The first lane was loaded with a DNA ladder, sizes were marked on
the image. The brightest band of 750 bp was about 100 ng and other bands about 50 ng. Lanes with correct sized amplified DNA were labeled. After PCR cloning, several bacterial clones were picked, grew into cultures,
and sent for Sanger sequencing. Then, we verified the sequencing results and used the correct ones for further experiments.
# Acquisition of twelve fusion protein DNAs
As mentioned above, we designed a total of twelve fusion proteins.
| Bst Pol 1: Bst-(G2S)3-albA1 | Bst Pol 2: albA1-(G2S)3-Bst |
| Bst Pol 3: Bst-(G2S)3-S.ssb | Bst Pol 4: S.ssb-(G2S)3-Bst |
| Bst Pol 5: Bst-(G2S)3-E.ssb | Bst Pol 6: E.ssb-(G2S)3-Bst |
| Bst Pol 7: Bst-(G2S)3-DbpA | Bst Pol 8: DbpA-(G2S)3-Bst |
| Bst Pol 9: Bst-(G2S)3-Sso10b | Bst Pol 10: Sso10b-(G2S)3-Bst |
| Bst Pol 11: Bst-(G2S)3-Sso7d | Bst Pol 12: Sso7d-(G2S)3-Bst |
To obtain the relevant DNA fragment corresponding to the fusion protein, we added homologous arms to both ends of the DNA fragments of the fusion protein and Bst Pol, thus enabling us to fuse these two DNA fragments into one complete fragment by overlap PCR.
Figure 11. Assembled fusion proteins, Bst Pol 1, Bst Pol 2, Bst Pol 3, Bst Pol 4, Bst Pol 5, Bst Pol 6. The first
lane was loaded with a DNA ladder, sizes were marked on the image. The brightest band of 750 bp was about 100 ng and other bands about 50 ng. Lanes with correct sized amplified DNA were labeled. After PCR cloning, several
bacterial clones were picked, grew into cultures, and sent for Sanger sequencing. Then, we verified the sequencing results and used the correct ones for further experiments.
Figure 12. Assembled fusion proteins, Bst Pol 7, Bst Pol 9, Bst Pol 11. The first lane was loaded with a DNA ladder, sizes were
marked on the image. The brightest band of 750 bp was about 100 ng and other bands about 50 ng. Lanes with correct sized amplified DNA were labeled. After PCR cloning, several bacterial clones were picked, grew into
cultures, and sent for Sanger sequencing. Then, we verified the sequencing results and used the correct ones for further experiments.
Figure 13. Assembled fusion proteins, Bst Pol 8, Bst Pol 10, Bst Pol 12. The first lane was loaded with a DNA ladder, sizes were marked on the image. The brightest band of 750 bp was about 100 ng and other bands about 50 ng. Lanes with correct sized amplified DNA were labeled. After PCR cloning, several bacterial clones were picked, grew into cultures, and sent for
Sanger sequencing. Then, we verified the sequencing results and used the correct ones for further experiments.
The result (Figure 3/4/5) shows a successful amplification of the DNA of interest. We performed gel extraction of the target region, obtain pure fusion protein DNA with homology arms for subsequent reactions. After ClonExpress® homologous recombination reaction, Fast-T1 competent bacteria were transformed with reactants and spread onto LB plates with ampicillin. We picked colony after 16 h culturing plates at 37℃. We further grow clones in 2 ml LB liquid medium with kanamycin, 37℃ shaking overnight.
Mini-prep was performed the following day, and the resulting plasmids were sent to Sanger sequencing, our synthesized DNA sequence was confirmed to be correct.